ITEMS FROM THE RUSSIAN FEDERATION

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.

Evaluation of drought resistance in spring wheat cultivars and lines at the seedling stage. [p. 107-108]

G.G. Vasilieva, N.V. Mironova, M.S. Naumova, and A.K. Glyanko.

Breeders in eastern Siberia see the creation of drought-resistant cultivars of spring wheat as an important objective. Climatic conditions of this region are characterized by drought in late May to June. During this period, spring wheat seedlings are at phase 3 (four leaves to tillering), which is why the ability of spring wheat plants to withstand soil moisture deficit without detriment to the harvest is one of the key characteristics of the cultivars grown in eastern Siberia. Our work aims to determine the drought resistance of a number of cultivars and lines of spring wheat by a method patented in Russia.

Material and methods. The studies were conducted on etiolated seedlings of spring wheat. Bloated wheat seeds were placed in cuvettes on filter paper wetted with tap water and grown in a thermostat at 27 C for 48 h. We then selected seedlings with identical primary root lengths. These seedlings were used as initial material for further work.

Test seedlings were placed in cuvettes for further growth on filter paper wetted with an osmotic (polyethyleneglycol) and kept in a thermostat at 27 C for 24 h. The seedlings then were thoroughly washed with water and kept on wet filter paper for 24 h at 27 C. Root length of the seedlings was measured and the rate of root growth (mm/h) calculated.

Control plants were grown under the same conditions on water. Based on the root growth speed in optimal conditions and after water stress, we determined the degree of root growth speed restoration after stress or action according to the formula: V test / V control E 100 %, where V is the seedling growth rate.

Results. The degree of growth after the impact produced by the stress (osmotic) proved to be directly proportional to the degree of plant resistance. We could split all the tested cultivars and lines into three groups; 1, high drought resistance (restoration of growth exceeds 100 %; 2, average drought resistance (from 90 to 100 %); and 3, low drought resistance (below 90 %) (Table 1). Cultivars and lines from the first group and some cultivars from the second group are of interest to breeders from the standpoint of their use in breeding new drought-resistant cultivars of spring wheat.

References.

• Patent 1734596 (Russia, priority of 08.06.1992). 1996. Method of evaluation of plants resistance to drought of northern and southern type at early stages of ontogenesis. Discoveries and Inventions, No 18.

The impact of spring frosts on the growth of spring wheat seedlings of different cultivars. [p. 108-112]

A.K. Glyanko, N.V. Mironova, and G.G. Vasilieva.

Introduction. Plant growth under various stress factors has been the subject of many studies, but the impact of spring frosts, which cause significant damage to crop production, have not been fully covered in the scientific literature (Shevelukha 1977; Drozdov et al. 1977; Musienko et al. 1986). The impact of spring frosts on plant growth is evaluated largely on the accumulation of fresh and dry matter by a plant and the final productivity. Nevertheless, these parameters do not allow the determination of fairly precisely such disturbances of growth processes, which take place during immediate cold impact and during several days right afterwards, these data being important for forecasting productivity of the plants subjected to frost. With this in view, our work targeted the assessment of dynamics of linear growth of leaves of various spring wheat cultivars immediately during cold stress and after several days.

Materials and methods. The following spring wheat cultivars from the collection of the N.I. Vavilov research institute of plant industry (St. Petersburg, Russian Federation) were used for the study: Milturum 553, Karola, Kzyl-Bas, Albidum 43, and Skala. These cultivars differ in the length of their vegetative period. Seeds of the cultivars were sown in pots with a capacity of 4 kg of dry sandy soil. A nutrient mineral mixture with a nitrate source of nitrogen was introduced according to Thomas et al. (1979). Soil moisture in the pots during plant growth was maintained at 60 % of the soil complete moisture capacity. Frosts were simulated in the Siberian phytotron (Irkutsk, Russian Federation) based on natural models. Temperature was measured automatically according to an established program (Kurets 1974). Three modeled frost types differed in the value of minimum temperature: -2 to -3 C, -4 to -5 C, and -6 to -7 C. In each variant, we identified three conventional periods of gradual decrease and increase in temperature: I - a temperature decrease from 20 to 0 C; II - a temperature decrease from 0 C to the minimal value and then increased to 0 C; and III - a temperature increase from 0 to 15 C (Glyanko and Mironova 1974). The duration of period II amounted to 6 h, of which for 2 h the temperature was decreased from 0C to a minimum value, for 1.5 h the plants were kept at the minimum temperature, and for 2.5 h the temperature was raised to 0C. During the frosts, the plants were kept in darkness. Before and after the frosts, plants were kept in the phytotron under stationary conditions of artificial illumination by DRL-700 lamps (illumination value 9,000 lux); a 24-h temperature of 19±1 C; and a 16-h illumination period. Linear growth of the upper leaf of the different spring wheat cultivars was measured by means of auxanographies (Shevelukha 1977). Seedlings were exposed to frost at the 4-leaf stage and growth was measured for 24 h. The data are presented in the form of average value of three independent experiments (M±SD).

Results and discussion. All the three experimental variants demonstrated significant growth reduction of different spring wheat plants exposed to below zero temperatures. Thus, at -2 to -3 C, leaf growth during 6 h in the different wheats varied from 0.96 (Kzyl-Bas) to 1.75 mm (Albidum 43), and growth rate from 0.16 to 0.29 mm/h, respectively. In the control plants, leaf growth varied from 4.5 to 9.0 mm and growth rate from 0.75 to 1.50 mm/h in Kzyl-Bas and Albidum43, respectively. Plants did not show any visual damage, proving the overcooling of seedlings during frosts.

Temperatures of -4 to -5 C inhibited leaf growth more intensely in Albidum 43 (0.84 mm), Milturum 553 (0.78 mm), Karola (0.72 mm), and Kzyl-Bas (0.66 mm), compared to the controls Albidum 43 (13.9 mm), Milturum 553 (9.0 mm), Karola (5.5 mm), and Kzyl-Bas (4.4 mm). The plants had visual damage. Albidum 43 and Milturum 533 plants had the lowest leaf damaged (in 55 and 10 % of plants, respectively), Skala plants were intermediate, and Karola and Kzyl-Bas plants showed no damage.

At -6 to -7 C minimum temperature, the seedlings showed practically no linear growth. Linear growth observed in two test variants at below zero temperature may apparently be accounted for by the increase of cells size due to ice formation in the tissues (Shevelukha 1977). There was observed visual damage of bottom leaves, which had terminated their growth, there were also cases of the whole plant perishing.

These data show that weak frosts (up to -4 to -5 C) in the period of immediate cold impact on the seedlings of different varieties of spring wheat did not fully stop growth processes, though inhibited them significantly. Frosts of -6 to -7 C almost completely inhibited linear growth of the leaf.

The aftereffect of frosts in nature creates a fairly favorable temperature regime. In our tests aftereffect of the frost was analyzed at the temperature increase from 0 C, with the plants being kept for 16 h in the cold chamber at the temperature, which in 4 h reached optimal value (15°C), and then they were transferred to the growth chamber with the environmental parameters identical to those preceding the frost.

It was found that immediately after the frost of -2 to -3 C the growth was intensely suppressed and its rate varied in different varieties from 0.13 ( Kzyl-Bas) to 0.37 mm/h (Milturum 553), and in control from 0.73 to 1.50 mm/h, respectively. These are average data for 16 h period and they do not reflect dynamics of the growth processes. With this in view it should be noted that within the first 1-1.5 h after the temperature increase up to 0 C linear growth practically completely stopped, and further (with the temperature increase) restored, but its intensity during 6 h remained considerably lower than in control (Figure 1).

After the plants transfer to the lit growth chamber (24 hours after the frost start) their growth in the test variant was slightly behind the growth in control (by 13-15 %). However, already in the darkness after the first 16 h light period test and control variants showed no difference, and in some case a little growth stimulation was observed. On the third day after the beginning of frost (in the light) dynamics of growth rate was analogous to that of control.

Thus, after effects of weak frosts did not produce significant negative impact on the growth processes. Growth inhibiting with the frost -4 to -5 C was observed only in the first 24 h, later growth inhibition was replaced for its stimulation and getting back to normal.

Frosts of -6 to -7 C caused intense damage, that is why we measured the growth of the plants, which had visually observed damage of leaves. Immediately after the frost termination (period III) the growth was considerably inhibited and its rate in different varieties amounted to 0.16-0.30 mm/h (Table 2). During the first 2-5 h after the temperature increase from 0°C the growth almost stopped (see Figure 1). This period observed even with the weak frosts may apparently called a 'cold shock' period, followed by the growth rate increase in the light, this rate still remaining below that of control plants. The most intense growth inhibition (up to 10 days) was observed in Albidum 43 (Table 2). Nevertheless, we note that the growth was measured in the plants with three perished leaves, and the growth stopped in the newly formed leaf, which was not there during the frost period.

In the light, the duration of growth inhibition of the plants subjected to the impact of -6 to -7 C frost increased. Thus, in Albidum 43, small inhibition of leaf growth in the darkness observed on the second and third day was followed by stimulation on the fourth day. Later, growth increase of test and control plants showed no differences.

Temperatures of -6 to -7 C distinctly demonstrated three phases of the unfavorable factor aftereffects: growth inhibition (the duration depended on the degree of the plant damage); growth stimulation (with little or no damage); and return to normal. The first phase may be subdivided into three subphases of growth inhibition, immediately after the frost termination, in light, and in darkness.

Immediately after frost termination, we observed the most intense inhibition of plants growth, which was accompanied by a complete stop in the first hours after the temperature increase above 0 C. In the light, these processes had different durations depending on the degree of plant damage and were shorter in the dark than in the light.

Artificial frosts obviously fail to fully reproduce natural frosts, which is why regularities of linear growth of leaves observed in the course of artificial frosts will differ from those in natural conditions (Vinter 1981). These differences are primarily conditioned by different environmental conditions (insolation intensity, air temperature, and humidity), to which the plants were exposed before, during, and after frost. In natural conditions, plant damage during the frost may be more significant, because high humidity and intense cooling of plant organs foster ice formation in their tissues, particularly in the case of radiation-type frosts.

In our experiments, the plants underwent weak and medium frosts, apparently in an overcooled state. Nevertheless, with frosts of any intensity the growth was undoubtedly inhibited in the first 24 h after its termination. Analogous data were obtained by other authors (Musienko et al. 1986), who demonstrated the most intensive inhibition of wheat seedlings growth in the first 24 h after the influence of cold temperature, regardless of its tension and duration. Further growth inhibition degree depended on the extent of plant damage.

The negative impact of light intensification on unfavorable factors on different physiological processes has been described in the literature and is a photodynamic effect (Ivanova et al. 1987). This is presumably caused by intensification of photooxidizing processes in chloroplasts due to activation of superactive radicals, in particular oxygen ones, formation of which is intensified in the light (Merzlyak 1989). All this may produce negative impact on the leaves growth during aftereffect of unfavorable factor.

The negative impact of light on the leaves linear growth was particularly well shown in the plants with intense damage. These data coincide with those of Ivanova et al. (1987), who noted that severe damage by high temperature on the plants getting more intense light during the functional repair stage. Consequently, successful repairing of disturbed physiological functions requires darkness. Rapid change in the illumination regime (darkness/light) also caused growth response in plants subjected to frost. In our experiments, exposure to light in the phytotron inhibited their growth for 15-20 min. Duration of growth stop with the change of illumination regime (darkness/light) on the first and second days after the frost termination amounted to 45-80 min depending on the frost intensity. Analogous photoactive responses were described earlier (Shevelukha 1986).

We established that a period of no growth immediately following cold impact termination (cold shock) is longer if the frost is more intense. Apparently, the process of plants adaptation to the damaging factor should be accompanied along with metabolism reconstruction by low intensity of growth processes (Tyurina 1960; Udovenko 1979; Trunova et al. 1987). It should be noted that in our experiments in non-adapted plants artificial frosts killed mostly bottom leaves, which terminated their growth. Intense frosts in the bushing phase damaged the whole plant with bushing sprouts undamaged. Consequently, the growing organs of wheat plants demonstrated the highest resistance. At the same time it should be noted that quick-ripening varieties (Albidum 43, Skala, vegetative period of 82­105 days) characterized by high intensity of growth processes were more intensely damaged than varieties with long vegetation period (Karola, Kzyl-Bas, 120-130 days). However, growth processes in the course of reparation period of quick-maturing wheat cultivars were characterized by higher intensity than those of late-maturing cultivars.

Growth stimulation observed in the repair period apparently witnesses small disturbances emerging in spring wheat seedlings under the influence of frost. Substitution of physiological functions inhibition by their stimulation is and index of reparation processes intensity, which confirms reversibility of damage and gradual restoration of the functions. To forecast the productivity of the plants subjected to frost, further research should be targeted at the identification of correlation between the degree of growth processes damage and productivity.

References.

• Drozdov SN, Sycheva ZF, Budykina NP, and Kurets VK. 1977. Ecological-physiological aspects of plants resistance to frosts. Nauka, Leningrad, 228 p. (In Russian).
• Glyanko and Mironova NV. 1974. Protein synthesis and nitrate-reductase activity in spring wheat leaves during the impact of spring frost on the plants. In: Questions of increase of efficiency of grain crops. Irkutsk, p. 134-138 (In Russian).
• Ivanova AP, Romanova TD, and Kurbskaya OB. 1987. Light impact on the disturbance of respiration by extreme temperature and on its further reactivation. In: Influence of physical and chemical factors on plants growth. Kazan, p. 55-63 (In Russian).
• Kurets VK. 1974. Irkutsk phytotron. Nauka, Novosibirsk, 96 p (In Russian).
  Merzlyak MN. 1989. Activated oxygen and oxidizing processes in plant cell membranes. Nauka, Moscow. V. 6, 403 p. (In Russian).
• Musienko NN, Daskalyuk TM, and Kaplya AV. 1986. Growth response of wheat seedlings to the impact of high temperatures. Plant Physiol 33:134-141 (In Russian).
  

Assessment of spring wheat cultivar competitiveness in the agrophytocenoses of East Siberia. [p. 112-114]

A.K. Glyanko, Sh.K. Khusnidinov (Irkutsk State Agrocultural Academy, Irkutsk, 664038, Russian Federation), T.G. Kudryavtseva, and G.G. Vasilieva.

Introduction. The agrophytocenoses of East Siberia (Russia) in the last 10­15 years have been intensely choked with weeds. This factor negatively affects the productivity of spring wheat and other crops cultivated in this region. Economic plants may successfully counteract weeds if they are highly competitive. Competitiveness is one of the three vital strategies of plants in accordance with the classification proposed by Ramensky (1938) and Grime (1979, 2001), which include competitors (C), stress-tolerators (S), and ruderals (R). These types of strategies (C, S, and R), as a rule, do not occur in nature in the pure state. More often plants have mixed strategy types (Hodgson et al. 1999). Economic plants, in the course of selection for economically valuable features (largely productivity), have to a significant extent lost the competitiveness and stress tolerance strategies and may be characterized as ruderal plants. The most important characteristics of R plants is their ability to respond quickly to an improvement in growth conditions via enhancement of growth, development, and an increase in productivity (Glyanko and Vasilieva 2002).

The soft wheats cultivated in East Siberia normally possess a high potential productivity (50­70 metric center (mc)/ha). Nevertheless, the actual productivity potential of these species equals 12­25 mc/ha. One reason for the decrease of spring wheat productivity in the East Siberian agrophytocenoses is low competitiveness of spring wheat species with weeds.

We found no scientific data on determination of competitiveness of spring wheat species, which is why we made an attempt to assess the ability of four spring wheat species to resist weeds in the field, in effect to identify their degree of competitiveness.

Material and methods. The four cultivars of soft spring wheat used for this study were Angara, Studencheskaya, Tulun 15, and Tulunskaya 12. The test was conducted on an ameliorated, light-gray forest soil. The test site area was 1 sq m. The tests were repeated four times. Test variants were randomized. Test sowings were performed on pure black fallow. The presowing treatment system included early spring harrowing, two presowing cultivations, and packing. The test schemes were variant 1, site without wheat (only weeds); variant 2, site with wheat sown with weeds removed during the vegetative period (wheat without weeds); and variant 3, site sown with wheat without the removal of weeds (wheat + weeds). The number of grains sown/m2 was 700 on 20 May, 2004. The plants were harvested manually at wax-ripeness (moisture 20-23 %) between 15-22 August, 2004.

The competitiveness of the spring wheat species and weeds was determined using the following formulas.

I. General competitiveness =

A1

K1 = ----------- , where

B1

K1 = general competitiveness, A1 = wheat productivity (straw and crop harvest) in variant 3 (wheat + weeds), and B1 = wheat productivity (straw and crop harvest) in variant 2 (wheat without weeds).

II. Productive competitiveness =

A2

K2 = -----------, where

B2

K2 = productive competitiveness, A2 = wheat grain productivity in variant 3 (wheat + weeds), and B2 = wheat crop harvest in variant 2 (wheat without weeds).

III. Weed competitiveness =

C

K3 = -----------, where

D

K3 = weed competitiveness, C = dry biomass of weeds in variant 3, and D = dry biomass of weeds in variant 1 (weeds without wheat).

Results. Quantitative and qualitative composition of weeds and their competitiveness. The estimate of weeds in variant 1 demonstrated that by 10 June on the site an average of 212 weeds were present, by 25 June 246 weeds, and by 7 July 405 weeds. The weeds were represented by 17 species with the following most predominant: Chenopodium album L., Senecio vulgaris L., Stelaria media L., Sonchus arvensis L., Cirsium oleraceum L., and also some species from the Panicum and Leguminosae families. Crepis testorum L., Spergula vulgaris L., Polygonum convolvulus L., Polygonum divaricatum L., Artemisia vulgaris L., and some other species occurred to a small degree.

The quantitative composition of weeds in the agrophytocenoses of different spring wheats showed that the dry mass of the weeds in agrophytocenoses with spring wheat at harvest were 15, 25, 51, and 116 g/m2 in Tulunskaya 12, Tulun 15, Studencheskaya, and Angara, respectively. Thus, weed growth was intensely oppressed in agrophytocenoses with spring wheats Tulun 15 and Tulunskaya 12. This conclusion is confirmed after determining the weed competitiveness coefficient K3. This parameter was the highest in agrophytocenosis with the spring wheat Angara (0.62), followed by Studencheskaya (0.27), Tulun 15 (0.13), and Tulunskaya 12 (0.08). The highest degree of weed competitiveness was observed in agrophytocenosis with Angara and the lowest with Tulunskaya 12.

Competitiveness of spring wheat cultivars. General and crop productivity were different in different cultivars (Table 3). Studencheskaya was characterized by the highest general and crop productivity, followed by Tulunskaya 12, Tulun 15, and Angara. Weed presence in the agrophytocenoses of spring wheat reduced general and crop productivity in all the cultivar to a different extent. The largest reduction in general productivity under the impact of weeds was observed in Tulunskaya 12 (by 22.5 mc/ha) and the lowest in Studencheskaya (by 5 mc/ha). Crop productivity under the impact of weeds was most reduced in Tulunskaya 12 (by 3.3 mc/ha) and Studencheskaya (by 3.2 mc/ha). In Angara and Tulun 15, the crop productivity was reduced by 2.4-2.5 mc/ha.

Calculation of competitiveness coefficients K1 and K2) of the spring wheats indicated that Tulunskaya 12 and Angara had the lowest values at 0.87 and 0.92 and 0.89 and 0.93, respectively. Tulun 15 had the highest values at 0.97 (K1) and 0.94 (K2); Studencheskaya insignificantly different at 0.95 (K1) and 0.93 (K2). Thus, we observed high sensitivity of weeds to Tulunskaya 12 (which shows in a low K3 coefficient of 0.08 and, on the other hand, a high sensitivity in this cultivar to weeds (K1= 0.87 and K2 = 0.92). Weeds respond less to Angara plants, which have a high K3 coefficient of 0.62 and low K1 and K2 coefficients (0.89 and 0.93, respectively).

Consequently, the reduction of productivity in Angara apparently is conditioned by the negative impact of weeds, that is interspecific competition for vital sources. In Tulunskaya 12, considerable importance apparently is the intraspecific competition associated with the density of wheat plants per square area unit. In conclusion, we note that the spring wheat cultivars studied possess various degrees of competitiveness with weeds. This property presumably depends both on interspecific and intraspecific competition between plants for vital sources.

References.

• Glyanko AK and Vasilieva GG. 2002. Vital strategies and their role in supporting the functional stability of spring wheat. Ann Wheat Newslet 48:124-125.
• Grime JP. 1979. Plant strategies and vegetation processes. J Wiley & Sons Ltd, Chichester, United Kingdom.
• Grime JP. 2001. Competitors, ruderals and stress-tolerators. J Wiley & Sons Ltd, Chichester, UK:
• Hodgson JG, Wilson PJ, Hunt R, Grime JP, and Thompson K. 1999. Allocating C-S-R plant functional types: a soft approach to a hard problem. Oikos 85:285-294.
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IThe influence of cyclosporin A, Ca+2 ions, and fatty acids on the mitochondrial swelling of cold-stressed and cold-hardening winter wheat shoots. [p. 114-116]

N.S. Pavlovskaya, O.I. Grabelnych, T.P. Pobezhimova, A.V. Kolesnichenko, N.A. Koroleva, and V.K. Voinikov.

Opening the high-conductance permeability transition pore (PTP) induces the mitochondrial permeability transition, which is characterized by mitochondrial swelling, uncoupling, and inner membrane permeabilization to solutes up to 1,500 Da. The opening of the PTP is an important factor in both necrotic and apoptotic cell death (Zoratti and Szabo 1995; Bernardi et al. 1998; Hirsch et al. 1998; Crompton 1999). PTP is considered a complex composed of a voltage-dependent anion channel (VDAC), an ADP/ATP antiporter, cyclophilin D, and possibly other proteins (Crompton 1999). He and Lemasters (2002) proposed a new model of PTP formation and regulation in which PTP forms by aggregation of misfolded integral membrane proteins damaged by oxidant and other stresses. The existence of classic cyclosporin A (CsA)-sensitive PTP in plant mitochondria still is discussed. PTP in plants is both sensitive to CsA (Arpagaus et al. 2002; Tiwari et al. 2002) and insensitive (Fortes et al. 2001; Curtis and Wolpert 2002; Virolainen et al. 2002). Induction of pore opening in plant mitochondria is accompanied by mitochondrial swelling and release of cytochrome c (Arpagaus et al. 2002; Curtis and Wolpert 2002; Tiwari et al. 2002; Virolainen et al. 2002). These events are characteristic for induction of animal PTP. The present investigation studies the mitochondrial swelling in the presence of inductors (Ca+2 and palmitic acid) and inhibitor (cyclosporin A) of PTP in cold-resistant winter wheat after short-term cold stress and cold hardening.

Materials and methods. Three-day-old etiolated seedlings of cold-resistant winter wheat cultivar Zalarinka were germinated on moist paper at 26°C. Seedlings were subjected to short-term (-1 C, 1 h) cold stress or were cold-hardened for 7 days at 4 C. The mitochondria were isolated from seedlings shoots by differential centrifugation (Pobezhimova et al. 2001). Isolated mitochondria were resuspended in 40 mM MOPS-KOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2. Mitochondrial swelling was followed spectrophotometrically by the decrease in absorbance of the mitochondrial suspension (0.25 mg/ml) under de-energized conditions at 26 C at 540 nm. We used two types of incubation media: 1) 300 mM sucrose and 20 mM MOPS (pH 7.4) and 2) 200 mM KCl and 20 mM MOPS (pH 7.4) (basic medium). Test reagents were used at concentrations of 1 mkM cyclosporin A, 1.75 mM Ca+2, and 50 mkM palmitic acid. In the experiments using CsA and Ca+2, the preincubation time was 5 min at 0 C. The concentration of mitochondrial protein was analyzed by according to Lowry et al. (1951). Results are represented as the mean of at least three determinations per experiment.

Results and discussion. Low and high temperatures and oxidative stress are known to be inductors of programmed cell death (PCD) in plants (Koukalova et al. 1997; Balk et al. 1999; Tiwari et al. 2002). In the present work, we studied the swelling of mitochondria, isolated from control (nonstressed and non-hardened), cold-stressed and cold-hardened winter wheat shoots.

We found that changes of optical density of mitochondrial suspension in the presence of sucrose did not occur (Figure 2g), whereas the isotonic KCl buffer caused a decrease of optical density and mitochondrial swelling (Figure 2a). This medium was used to study the influence of mitochondrial pore inductors and inhibitors on the swelling winter wheat mitochondria in our work.

In experiments with incubation of control winter wheat mitochondria with CsA, we detected a decrease in optical density of mitochondrial suspension (Figure 2b). Because Ca+2 accumulation in mitochondria is known to cause PTP opening (Gunter and Pfeiffer 1990), we studied the influence of Ca+2 on the swelling of winter wheat mitochondria that were preliminarily incubated with and without CsA. The presence of Ca+2 in the incubation medium stimulated the extent of swelling already in 20 sec of incubation, and this stimulation was 3-fold in 5 min (Figure 2c), compared with the swelling of mitochondria incubated without Ca+2. The Ca+2-induced swelling was inhibited after preliminary incubation of mitochondria with CsA. In this case the swelling was sensitive to CsA action on 70-75 % (in 5 and 10 min of incubation) (Figure 2d). We observed an increase in mitochondrial swelling extent in the presence other inductor of PTP - saturated fatty acid - palmitic acid, the action of which was similar to Ca+2 action. Palmitic acid caused a 4-fold increase in swelling within 5 min of incubation, which was sensitive to CsA addition (Figure 2e and f).

Short-term exposure by low temperature on the winter wheat shoots caused the decrease of optical density in isolated mitochondria and theirs swelling. The swelling extent in 5 min of incubation of the mitochondria was 1.4 times higher than the swelling extent of mitochondria isolated from nonstressed shoots (Figure 3a). The swelling of mitochondria from cold-stressed shoots was less sensitive to CsA (20 % and 40 % inhibition in 5 and 10 min of incubation with this inhibitor, respectively) (Figure 3b). Incubating mitochondria with Ca+2 stimulated the extent of swelling, which was 2.75 times higher (in 5 min of incubation) than the swelling of mitochondria incubated without Ca+2 (Figure 3c). We detected that CsA did not inhibit the Ca+2-induced swelling but stimulated swelling (70 % increase in 5 min of incubation) (Figure 3d). The swelling induction of mitochondria from cold-stressed shoots in the presence of palmitic acid was less expressed (2 times) in comparison to that of the acid in mitochondria isolated from nonstressed shoots. Palmitate-induced swelling of mitochondria from cold-stressed shoots was fully inhibited by CsA (Figure 3e and f).

Cold hardening of winter wheat shoots caused the swelling of isolated mitochondria similar with swelling of isolated mitochondria from cold-stressed shoots. The swelling extent of mitochondria was 1.3 times as higher (in 5 min of incubation) than that of mitochondria isolated from nonstressed shoots (Figure 4a). The swelling of mitochondria of cold-hardened shoots was less sensitive to CsA (20 % inhibition in 5 and 10 min) (Figure 4b). Incubating mitochondria with Ca+2 stimulated 2-fold degree of swelling during the time of incubation in comparison with mitochondria incubated without Ca+2 (Figure 4c). Ca+2-induced swelling was not sensitive to CsA (Figure 4d). The swelling induction in mitochondria isolated from cold-hardened shoots after the addition of palmitic acid was less expressed and 1.4-fold compared with mitochondria that were incubated without the addition of the acid. Palmitate-induced swelling of mitochondria from cold-hardened shoots was fully inhibited by CsA (Figure 4e and f).

Mitochondrial swelling is one event of PTP opening. We concluded that the swelling of winter wheat mitochondria are associated with opening of mitochondrial pore. The stimulation of swelling by Ca+2 and the inhibitory effect of CsA indicate Ca+2-dependent, CsA-sensitive, mitochondrial pores function in winter wheat shoots. At the same time, cold stress and cold hardening decrease the sensitivity of mitochondria to cyclosporin A, which may function in CsA-insensitive pores in conditions of cold stress and hardening. Our data show that different mechanisms of opening pore regulation in normal and stress conditions exist.

Acknowledgments. This work was performed, in part, with the support of the Russian Science Support Foundation, Russian Foundation of Basic Research (project 05-04-97231), and Siberian Division of Russian Academy of Sciences Youth Grant (project 115).

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The influence of oxidative stress on winter wheat mitochondria function and alternative oxidase contribution. [p. 117-119]

O.I. Grabelnych, T.P. Pobezhimova, N.Yu. Pivovarova, N.S. Pavlovskaya, N.A. Koroleva, A.V. Kolesnichenko, and V.K. Voinikov.

Oxidative stress has significant influence on cell metabolism of plants. Under oxidative stress conditions the expression of such mitochondrial protein as cyanide-resistant alternative oxidase (AOX) occurs (Maxwell et al. 1999; Szal et al. 2003; Polidoros et al. 2005). AOX catalyzes quinol-oxygen oxidation/reduction that is not linked to proton pumping and consequently does not generate a proton electrochemical gradient. The low expression of AOX in roots of wheat seedlings can explain observed symptoms of oxidative stress (Biemelt et al. 1998). One of the functions of alternative oxidase is its antioxidant role (Popov et al. 1997; Purvis, 1997; Maxwell et al. 1999). AOX induction can represent the important mechanism prevented activation of programmed cell death (Amor et al. 2000; Robson and Vanlerberghe 2002; Vanlerberghe et al. 2002).

We have shown previously that cold shock and cold hardening caused the increase of AOX contribution to total respiration of winter wheat mitochondria (Grabelnych et al. 2004). Taking into account the important role of alternative oxidase in plants, we studied the reaction of winter wheat mitochondria under oxidative stress conditions and AOX activity changes. The aim of the present investigation was to study influence of oxidative stress on winter wheat mitochondria energetic activity and AOX contribution to total respiration of the mitochondria in these conditions.

Materials and methods. Three-day-old etiolated shoots of winter wheat (Triticum aestivum L, cv. Zalarinka) germinated on moist paper at 26 C, were used. Oxidative stress was induced by immersing root tips of intact three-day-old seedlings in 0.5 mM solution of H2O2 in the dark at 26 C for 3 h. Coleoptiles of treated seedlings were harvested for mitochondria isolation.

Mitochondria were extracted from winter wheat shoots by differential centrifugation as describes previously (Pobezhimova et al., 2001). Isolated mitochondria were resuspended in 40 mM MOPS-KOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2. The intactness of mitochondria was determined by and measurement of cytochrome c oxidase activity and swelling measurement. The activity of mitochondria was recorded polarographically at 27 C using a platinum electrode of a closed type in a 1.4 ml volume cell. The reaction mixture contained 125 mM KCl, 18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. Oxidations substrates were 10 mM Malate in the presence of 10 mM glutamate and 8 mM succinate in the presence of 10 mM glutamate. During succinate oxidation, 3 mkM rotenone, which blocks electron transfer through complex I, was added to incubation medium. Test reagent concentrations were 1 mM benzohydroxamic acid (BHAM) (AOX inhibitor), 20 mkM antimycin A (complex III inhibitor), and 0.4 mM KCN (complex IV inhibitor). Polarograms were used to calculate the rates of phosphorylative respiration (state 3), nonphosphorylative respiration (state 4), the rate of respiration after BHAM addition, the rate of respiration after Ant-A addition, respiration control by Chance-Williams, and the ADP:O ratio (Estabrook 1967). The concentration of mitochondrial protein was analyzed by Lowry method (Lowry et al. 1951). All the experiments were made in 3­6 preparations. The data obtained were analyzed statistically, i.e., arithmetic means and standard errors were determined.

Results and discussion. Winter wheat mitochondria isolated from shoots exposed under oxidative stress differ significantly in their coupling degree of oxidation and phosphorylation processes and phosphorylative and non-phosphorylative rates from nonstressed shoots mitochondria. In malate-oxidizing (when transfer of electrons starts with complex I of respiratory chain) winter wheat mitochondria, oxidative stress caused a 56.2 % decrease of phosphorylative and a 23.1 % decrease of nonphosphorylative rates of respiration as comparison to nonstressed mitochondria (Figure 5). At the same time, we observed a 43.0 % decrease of respiratory control coefficient under stress conditions. When succinate was used as oxidation substrate (when transfer of electrons starts with complex II of respiratory chain), oxidative stress caused a 52.7 % decrease of phosphorylative, a 54.0 % decrease of nonphosphorylative rates of respiration, and a 20.0 % decrease of respiratory control coefficient in winter wheat mitochondria (Figure 6). The decrease of respiratory rates points out on the repression oxidative phosphorylation under oxidative stress.

Using inhibitor analysis, which allows the blocking of terminal oxidases or certain electron-transport chain complexes, we studied the contribution of different electron transport pathways into total plant mitochondria oxygen uptake. Changes of cytochrome and alternative pathways contribution to respiration of winter wheat mitochondria under oxidative stress were investigated. The contribution of different electron transport pathways to respiration of winter wheat mitochondria from nonstressed shoots during malate oxidation is that the main part (62.5 %) of mitochondrial oxygen uptake depends on the cytochrome pathway function (Figure 5). AOX contribution was about 12.5 % and residual respiration was about 25.0 % (Figure 5). Oxidative stress caused an increase in the contribution of the AOX pathway in oxygen uptake that was about 52.6 % and a decrease in the contribution of the cytochrome pathway of about 31.2 % (Figure 5).

Succinate-oxidizing winter wheat mitochondria showed a similar picture. We found that the main part (85.6 %) of mitochondrial oxygen uptake in nonstressed mitochondria depends on the cytochrome pathway function (Figure 6). Oxidative stress caused the increase of contribution of AOX pathway in oxygen uptake of about 41.2 %, and the decrease in the contribution of the cytochrome pathway of about 42.0 % (Figure 6). Residual respiration was about 14.9 %.

Oxidative stress induced by H2O2 causes a decrease in the coupling degree of oxidative phosphorylation in winter wheat mitochondria and the increase of alternative oxidase activity. From this data on alternative oxidase activation in winter wheat mitochondria under oxidative stress conditions and previous data obtained about the significant increase of AOX contribution to total respiration of winter wheat mitochondria under cold shock and cold hardening (Grabelnych et al. 2004), we identify the protective role of this protein in stress conditions.

Acknowledgments. The work has been performed, in part, with the support of the Russian Science Support Foundation, Russian Foundation of Basic Research (project 05-04-97231) and Siberian Division of Russian Academy of Sciences Youth Grant (project 115).

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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.

Defensins of Triticum kiharae and other wheat species. [p. 119-121]

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).

Fungal and bacterial diseases cause severe crop damage and present an ongoing challenge for farmers. Breeding crops resistant to multiple infections by conventional methods has serious limitations. Introduction of antimicrobial peptides via genetic transformation of plants offers a strategy for production of resistant crops. Several antimicrobial peptides expressed in plants demonstrated enhanced resistance to pathogens (Allan et al. 2004).

Defensins are small cationic peptides implicated in the first-line host defense against pathogens. Their spatial structure is similar in different species including animals and plants and involves Cys-stabilized a/b-motif. Despite striking similarity in tertiary structures, their amino-acid sequences are highly variable except for eight cysteine residues that are conserved (Thomma et al. 2002). Variation in primary structure may account for different biological functions reported for defensins. They were found to exert antifungal (Terras et al. 1995), antibacterial (Segura et al. 1998), and inhibitory activities against a-amylases (Bloch and Richardson 1991) and proteinases (Wijaya et al. 2000). Increasing evidence indicates that the activities of defensins within a given species may be different. Of two defensins isolated from Medicago truncatula, MsDef1 strongly inhibited the growth of F. graminearum in vitro, whereas MsDef2, which shares a 65 % amino-acid sequence homology with MsDef1, was inactive against F. graminearum (Spelbrink et al. 2004). Similar results were obtained earlier with Raphanus sativus defensins (De Samblanx et al. 1997). Unlike the mammalian defensins, antifungal plant defensins cause membrane permeability through specific interaction with high-affinity binding sites on fungal cells (Thevissen et al. 2000) but do not form ion-permeable pores in artificial lipid bilayers. Defensins likely may act through different mechanisms. Defensins from Zea mays inhibited sodium currents in a rat tumor cell line but showed no antifungal activity (Kushmerick et al, 1998). A plant defensin, MsDef1, selectively blocked the mammalian L-type Ca+2 channel, however, two structurally similar defensins, MsDef2 and Rs-AFP2 from Raphanus sativus, did not block the L-type Ca+2 channel (Spelbrink et al. 2004).

Our previous studies showed that T. kiharae, a synthetic allopolyploid produced by crossing T. timopheevii subsp. timopheevii with Ae. tauschii and is highly resistant to most fungal pathogens, is a good source of antimicrobial peptides and a promising model for studying their properties and role in plant defense. Earlier we identified seven families of antimicrobial peptides in the seeds of this species. Here, we focus our attention on defensins and compared their structure and complexity with defensins from other Triticum species.

Material and methods. Seeds of several wheat species were used in this study: T. kiharae, T. turgidum subsp. timopheevii, T. militinae, and T. aestivum subsp. aestivum cultivars Khahasskaya and Rodina). Wheat flour was defatted with petroleum ether (1:10) and extracted with an acid solution (1 M HCl and 5 % HCOOH) for 1 h at room temperature and desalted on a Aquapore RP300 column. Freeze-dried acidic extract was subjected to chromatography on Heparin Sepharose. Proteins and peptides were eluted with a stepwise NaCl gradient. The 100-mM NaCl fraction was collected, desalted as described above and separated 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. Mass spectra were acquired on a model Reflex III mass spectrometer (Bruker Daltonics, Bremen, Germany). 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. From T. kiharae seeds, six defensins were isolated from the 100-mM fraction. Their N-terminal amino acid sequences were as follows:

D1: RTCQSQSHKFKGAC
D2: RTCESQSHKFKGPCF
D3: RDCKSDSHKF
D4: RDCTSQSHKFVG
D5: RECRSESKKF
D6: RDCRSQSKTFVG

Sequence comparison showed that the purified peptides were highly homologous and represented a family of closely related peptides differing in point amino acid substitutions. The molecular masses of defensins determined by mass spectrometry were 5,735, 5,691, 4,970, 4,980, 5,150, and 5,089 Da for D1 to D6, respectively. The total yield of these peptides estimated from the results of sequencing averaged approximately from 0.2 to 2.4 g/g of dry seed and comprised 0.07 % of the total protein. We determined the number of cysteine residues in defensins by estimating mass difference between alkylated and nonalkylated proteins because their position and number is a characteristic feature of this class of antimicrobial peptides. The results obtained for all peptides were similar indicating the presence of eight cysteine residues.

The RP-HPLC separation of defensins from other species produced very similar chromatographic profiles, suggesting that homologous peptides occur in all species studied. However mass determination and N-terminal sequencing were used to confirm this suggestion. The results obtained showed that D1­D6 defensins were present n all species studied. For example in the T. aestivum subsp. aestivum cultivar. Khakasskaya, the molecular masses of the defensin-like peptides were 5,736, 5,692, 4,971, 4,981, 5,151, and 5,090, which is the same as we obtained for T. kiharae, indicating the presence of identical peptides. Mass data were confirmed by N-terminal sequencing.

Homologous peptides were isolated from other wheat species, however, the amount of individual peptides varied in different species indicating that their expression level in seeds may be different. Variation in expression level may account for differences in the resistance level and/or specificity of reaction to the pathogen attack. Our results show that defensin complement in wheat species (T. timopheevii subsp. timopheevii, T. militinae, and T. aestivum subsp. aestivum) of different genomic composition (AbG, for T. timopheevii subsp. timopheevii and T. militinae, AbGD for T. kiharae, and AuBD for T. aestivum subsp. aestivum) is similar, therefore, the genes encoding D1-D6 defensins were already present in tetraploid wheat and were preserved in the evolution during the formation of hexaploid species.

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