GrainGenes

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.

 

The impact of early autumn frost on the efflux of nitrous substances in spring wheat. [p. 119-122]

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

Introduction. Frost during the period of ripening and maturing of spring wheat grain is a typical natural phenomenon in many parts of the world (Russian Federation, Scandinavian countries, Canada, and the USA). The short-term impact of negative temperature on plants at this time (particularly during milky ripeness) is a decrease in the technological and sowing qualities of the grain resulting in the formation of so-called 'frost-beaten grain' with low germinating capacity, low test weight, and low flour production (Lamb 1967; Razumovsky and Zabotina 1969; Reimers and Illi 1974). Wheat from such grain have a low initial intensity of growth and morphological distortions (Pavlov 1967; Illi et al. 1979). Such properties of the grain after an early autumn frost are apparently conditioned by the negative influence of cold on the physiological-biochemical processes that are responsible for grain ripening and maturing. Among these processes, those of nitrous substances efflux from vegetative parts of the plants to reproductive organs in the period of kernel formation are very important. The present work was aimed at determining the degree of impact of early autumn frost at milky ripeness on the nitrogen efflux from leaves, stems, and spike (chaff) towards the kernel.

Materials and Methods. The spring wheat Skala was grown in a climatic chamber with the temperature regime of 19 ± 1/1 5 ± 1 C (day/night). Illumination was by incandescent lamps DRL-700 with a light intensity of 14 ± 0.5 kl and a 16-hour light period. Infrared radiation of the lamps was precluded by a water screen. Sandy soil with a low content of total nitrogen (0.009 %) was used for plant growth. Macroelements from Gelrigel's mixture and microelements as per Hogland (Grodzinsky and Grodzinsky 1973) were introduced in the enameled vessels with soil (tank capacity was 9 kg of dry soil). Soil moisture was maintained at 70 % of complete soil moisture capacity (SMC). Ten days prior to termination of the experiment, soil moisture was reduced to 45-50 % SMC. Each vessel contained five plants at the end of the experiment.

The plants were subjected to an artificial frost with the intensity of -6.5 ± 0.5 C at milky ripeness stage (moisture 55-60 %). The frost was simulated at night, in the dark. All temperature regulating operations in the freezer were performed automatically in compliance with the program, which included a gradual reduction of temperature from 15 to 0 C (1 C/12 min). Further reduction down to a minimum negative temperature of -6.5 ± 0.5 C was at 1 C/22 min (Kurets 1974). After 1.5-h exposure at -6.5 ± 0.5 C, the temperature was increased to 0°C and then to 15 C with a speed 1 C/12 min. Total duration of negative temperature impact on the plants was 6 hours. The plants did not show any visible damage.

Control plants were placed in the chamber with optimal growth temperature. One and one-half hours after the termination of extreme temperature factor impact on the test plants, both sets of plants were fed via roots with 15N-labeled Ca(NO3)2 at a dose of 100 mg of nitrogen/vessel. Nitrogen enrichment was 95.05 atomic % 15N. Further growth of both control and test plants was conducted at the temperature conditions preceding the frost.

Samples for analysis were selected after 7 days (beginning at waxy ripeness phase, grain moisture 38-40 %) and 19 days (end of waxy ripeness phase, grain moisture 17-19 %). The plants were divided into leaves, stems, shaft, and grain. After drying at 105°C, the samples were reduced to a powder. Plant material samples were burned in concentrated sulfuric acid with the catalyst (selenium). Nitrogen was determined per K'eldal's micromethod (Ermakov et al. 1987). The samples were enriched with heavy-isotope nitrogen on an MI-1305 mass-spectrometer (Optical-mechanical Plant, Sumy, Ukraine). The content of marked nitrogen in the plants samples was calculated according to the formula of isotope dilution (Korenkov 1977). The test was repeated six times. The results are presented as average arithmetic means with the standard error identified. Reliability of differences was assessed by Student criterion.

Results and Discussion. Depending on the state in which plants overcome the impact of negative temperature, supercooling or ice formation in the cells, the degree of damage will vary (Drozdov et al. 1977). Our test wheat plants went through the artificial frost apparently in the state of supercooling, because they showed no visible damage. The absolute nitrogen content in wheat kernel of the control constantly increased until the end of plant growth (Table 1). In the variant with frost, we observed a tendency towards reduction of nitrogen content in the kernel, from the end of the waxy ripeness phase through complete kernel ripeness. Relative nitrogen content in the kernel (% of dry mass) increased in both test variants by the end of waxy ripeness phase and was reduced at complete kernel ripeness. However, this reduction was statistically reliable only in the variant with frost (P > 0.95).

Table 1. Change in the nitrogen content (mg/plant) in different organs of spring wheat plants under the impact of early autumn frost. * numerator = mg/plant; denominator = % from dry weight of grain.

Treatment	Start of waxy ripeness stage			End of waxy ripeness stage			Complete grain ripeness
	Total	Excess atom 15N %	Marked	Total	Excess atom 15N %	Marked	Total	Excess atom 15N %	Marked
Leaves
Control	7.5 ± 0.30	5.7 ± 0.30	0.5 ± 0.02	5.7 ± 0.30	2.6 ± 0.20	0.2 ± 0.01	4.9 ± 0.10	2.9 ± 0.20	0.2 ± 0.01
Early frost	8.4 ± 0.50	6.4 ± 0.40	0.6 ± 0.02	4.8 ± 0.20	3.8 ± 0.20	0.2 ± 0.01	4.8 ± 0.20	4.7 ± 0.40	0.2 ± 0.01
Stems
Control	22.7 ± 1.00	9.1 ± 0.05	2.2 ± 0.10	12.9 ± 0.60	6.8 ± 0.10	0.9 ± 0.01	10.8 ± 0.60	6.9 ± 0.10	0.8 ± 0.04
Early frost	19.3 ± 0.80	10.6 ± 0.50	2.2 ± 0.20	7.8 ± 0.40	4.6 ± 0.20	0.4 ± 0.01	7.1 ± 0.40	6.6 ± 0.05	0.5 ± 0.02
Chaff
Control	7.0 ± 0.30	7.2 ± 0.10	0.5 ± 0.03	3.1 ± 0.10	5.8 ± 0.10	0.2 ± 0.01	2.2 ± 0.05	6.8 ± 0.30	0.2 ± 0.01
Early frost	5.2 ± 0.05	6.2 ± 0.05	0.3 ± 0.02	2.7 ± 0.20	4.8 ± 0.30	0.1 ± 0.01	2.2 ± 0.05	6.8 ± 0.30	0.2 ± 0.01
Grain
Control	28.5 ± 0.60* 2.2 ± 0.10	17.2 ± 0.70	5.2 ± 0.20	46.7 ± 1.00 3.3 ± 0.05	18.1 ± 0.90	8.9 ± 0.40	51.5 ± 0.90 3.0 ± 0.10	17.2 ± 0.50	9.3 ± 0.30
Early frost	22.4 ± 0.90 2.2 ± 0.05	13.7 ± 0.30	3.3 ± 0.10	51.0 ± 1.00 3.5 ± 0.10	14.0 ± 0.80	7.6 ± 0.40	47.1 ± 1.30 2.9 ± 0.05	16.6 ± 0.50	8.3 ± 0.30

With ripening and maturing of kernels, the total nitrogen content in leaves, stems, and shaft was reduced (Table 1). Nevertheless, in the period from the end of waxy ripeness until complete kernel ripeness, the frost variant demonstrate an increase in excessive 15N % and marked nitrogen content in stems and shaft. In the leaves, analogous changes in these parameters were statistically unreliable. The rise of marked nitrogen content in the vegetative organs apparently was related to the absorption of nitrogen introduced in the soil during fertilization.

The highest nitrogen content in the kernel from the stems by complete kernel ripeness was observed in both control (62 %) and treated (65 %) plants (Table 2). The leaves had 13 % (control) and 19 % (treated) and the shaft 25 % (control) and 16 % (treated) total stored nitrogen. Similar results characterized the period from the beginning to end of waxy ripeness. We concluded that during both interphase periods, frost resulted in a reliable reduction of nitrogen efflux from shaft to kernel (P > 0.99). We also observed a reliable (P > 0.95) increase in nitrogen efflux from the leaves during the period from beginning to end of waxy ripeness. Nevertheless, these changes were unreliable for the beginning of the waxy ripeness phase.

Table 2. Change in total and marked nitrogen efflux from vegetative organs of spring wheat under the impact early autumn frost. Numerator = mg/plant; denominator = % of total nitrogen.

Treatment	Efflux of total nitrogen				Efflux of marked nitrogen
	Total	from leaves	from stems	from chaff	Total	from leaves	from stems	from chaff
Start to end of waxy ripeness phase.
Control	15.5 ± 1.3 100	1.8 ± 0.4 12	9.8 ± 1.2 63	3.9 ± 0.3 25	1.9 ± 0.1 100	0.3 ± 0.02 16	1.3 ± 0.10 68	0.3 ± 0.03 16
Early frost	17.6 ± 1.7 100	3.6 ± 0.5 21	11.5 ± 0.9 65	2.5 ± 0.2 14	2.4 ± 0.2 100	0.4 ± 0.02 17	1.8 ± 0.20 75	0.2 ± 0.01 8
Start of waxy ripeness to complete ripeness.
Control	19.3 ± 1.2 100	2.6 ± 0.3 13	11.9 ± 1.2 62	4.8 ± 0.3 25	2.0 ± 0.1 100	0.3 ± 0.05 15	1.4 ± 0.10 70	0.3 ± 0.02 15
Early frost	18.8 ± 1.0 100	3.6 ± 0.5 19	12.2 ± 0.9 65	3.0 ± 0.1 16	2.2 ± 0.2 100	0.4 ± 0.02 18	1.7 ± 0.20 77	0.1 ± 0.02 5

Further data analysis showed that an increase in nitrogen content in the kernel of the plants subjected to frost by complete ripeness amounted to 24.7 ± 1.6 mg/plant (range 22.4 ± 0.9-47.1 ± 1.3 mg/plant), including marked nitrogen 5.0 ± 0.3 mg/plant (range 3.3 ± 0.1-8.3 ± 0.3 mg/plant) (Table 1). Consequently, an increase in total and marked nitrogen content in the kernel exceeded the amount of nitrogen arriving from the vegetative organs by 24 % (P > 0.95) and 57 % (P > 0,99 for total and marked nitrogen, respectively (the decrease in total and marked nitrogen from vegetative organs amounted to 18.8 ± 1.0 and 2.2 ± 0.2 mg/plant, respectively). In other words, plants subjected to frost used both nitrogen accumulated in the vegetative organs and nitrogen from soil solution for kernel formation, a considerable part of which was marked nitrogen.

The control plants showed similar regularities but used soil nitrogen to lesser extent. Thus, the total nitrogen increase in the kernel exceeded its efflux from vegetative organs by 16 % (the difference is unreliable) and marked nitrogen increased to 51 % (P > 0.99).

If we calculate the value of parameters considered in relation to the period beginning to the end of waxy ripeness, we find that total nitrogen inflow into the kernel will exceed its efflux from the vegetative organs by 38 % (11.0 ± 2.1 mg/plant; P > 0.99) in the frost variant and 15 % (2.7 ± 18 mg/plant; the difference is unreliable) in control plants. Consequently, between the beginning and end of waxy ripeness, nitrogen from the soil also was used for kernel formation.

In the plants subjected to frost, use of total nitrogen from soil for kernel formation from the end of waxy ripeness to complete ripeness declined from 38 % (11.0 ± 2.1 mg/plant) to 24 % (5.9 ± 1.9 mg/plant) (the difference is unreliable) and that of marked nitrogen increased from 44 (1.9 ± 0.1 mg/plant) to 57 % (2.8 ± 0.2 mg/plant) (P > 0.95). We conclude that frost stimulated the use of marked nitrogen for kernel formation. However, frost did not produce a reliable impact on the total nitrogen content in the kernel at maturity (Table 1). We note a reduction in the relative content of total nitrogen in the kernel from the end of waxy ripeness to complete ripeness (Table 1). Nitrogen loss from the kernel at maturity has been described (Kumakov 1987; Korovin 1984). Korovin (1984) indicated that this phenomenon may be accounted for by increased moisture and low environmental temperature. The concrete physiological-biochemical mechanism of this fact is unknown.

Our tests on early autumn frosts produced a significant impact on the efflux of total and marked nitrogen from the vegetative organs to the kernel. Total nitrogen efflux from the leaves increased and from the shaft was reduced. We observed an increased of marked nitrogen efflux from the stems between the beginning and end of the waxy ripeness stage. Stimulating or hampering physiological functions after stress may be connected with the ability of the plant and depend on the character and intensity of the stress (Drozdov at al. 1977; Al'tergot 1981). For the spike rachis and the closeness of the nitrogen source used by maturing kernels, then a reduction in nitrogen after a frost could impact protein synthesis in the kernel. Earlier investigations showed that an early autumn frost causes changes in the proportion of vital reserve proteins of spring wheat kernels (glutenin and gliadin) (Glyanko and Trufanov 1999). Similar changes may affect baking qualities of wheat grain.

References.

• Al'tergot VF. 1981. Increased temperature impact on the plants in experimental and natural conditions. Nauka, Moscow (in Russian).
• Drozdov SN, Sycheva ZF, Budykina NP, et al. 1977. Ecological-physiological aspects of plants resistance to frost. Nauka, Leningrad (in Russian).
• Ermakov AI, Arasimovich VV, and Jarosh NP. 1987. Methods of biochemical investigation of plants. VO Agropromizdat, Leningrad (in Russian).
• Glyanko AK and Trufanov VA. 1999. Nitrogen accumulation and protein content in spring wheat corn under the influence of frost. Agric Biol 5:36-43 (in Russian).
• Grodzinsky AM and Grodzinsky DM. 1973 . Concise manual on plant physiology. Naukova Dumka, Kiev (in Russian).
• Illi IE, Orekhova GV, and Lysak LN. 1979. Temperature impact on the dynamics of proteins of wheat seeds in the stage of formation. In: Biochemical and physiological investigations of the seeds. (Reimers FE Ed). Irkutsk. Pp. 42-57 (in Russian).
• Korenkov D. 1977. Methods of application of nitrogen isotope 15N in agricultural chemistry. Kolos Publishing House, Moscow (in Russian).
• Kumakov VA. 1978. Biology of spring wheat. In: The spring wheat. Kolos Publishing House, Moscow. Pp 27-72 (in Russian).
• Korovin -I. 1984. Plants and extreme temperatures. Hydrometeoizdat Publishing House, Leningrad (in Russian).
• Kurets V. 1974. The Irkutsk phytotron. Nauka, Novosibirsk (in Russian).
• Lamb C.A. 1967. Physiology of wheat. In: Wheat and Wheat Improvement (Reitz LP and Quisenberry KS, Eds). Amer Soc Agron, Madison, WI. Pp. 199-249.
• Pavlov AN. 1967. Protein accumulation in wheat and maize grains. Nauka, Moscow (in Russian).
• Razumovsky AG and Zabotina EI. 1969. Sowing and technological qualities of frost-beaten corn. In: Materials of Krasnoyarsk SRI of Agriculture. 5:18-22 (in Russia).
• Reimers FE and Illi I.E. 1974. Physiology of Siberian cultural plants seeds. Nauka, Novosibirsk (in Russian).
  

 

Influence of low-intensity laser radiation on lipid peroxidation in wheat callus cultures. [p. 122-124]

R.K. Salyaev, L.V. Dudareva, S.V. Lankevich, V.M. Sumtsova, and E.G. Rudikovcka.

During the last two decades, low-intensity laser radiation in the visible and near-infrared ranges is finding an increasing application in biological studies including biotechnological research (Grishko et al. 1999). However, up to now, the data are scarce on the mechanism of the effect of laser light on biological subjects because of difficulties encountered in analyzing light energy transformations in cells and the complexity of the response of a complex living system, multilevel in its organization, to laser effects. At present, we are actively pursued the effect of low-intensity laser radiation on animal and human tissues (Skobelkin 1997). At the same time, we emphasize that plants are evolutionary more adapted to the perception and assimilation of light energy, because they include a multitude of light-sensitive compounds, such as phytochromes and photosynthetic pigments. In our opinion, plants are particularly interesting for investigating the mechanisms of laser radiation.

Previously, we showed that low-intensity laser radiation stimulates morphogenetic processes in tissue cultures of wheat and wild grasses, such as rhizogenesis and the formation of morphogenic calli and regenerated plants (Salyaev 2001a and b). However, laser light can be regarded not only as a stimulant, but also as a stress agent damaging cells and tissues, most notably at the initial stages of radiation (Rogatkin and Chernyi 1999).

Generation of reactive oxygen species and an enhancement in lipid peroxidation processes are known to be the primary responses to many stress agents (Baraboi 1991; Smirnoff 1993; Kurganova et al. 1997; Kuznetsov 2000). In this connection, establishing whether or not the low-intensity laser radiation can induce lipid peroxidation in plant tissues was important. An analysis of accumulation of the primary and secondary products of lipid peroxidation can be regarded as one of possible lines of approaching such problem. Therefore, the objective of this work was to investigate the effect of low-intensity laser radiation on the process of accumulation of secondary lipid peroxidation products in wheat tissue culture.

The wheat cultivar Skala (bred in Siberia) tissue culture was used in this work. A mature embryo with half of endosperm was used as an explant. Callus formation was induced on a modified Murashige-Skoog medium complemented with 2 % sucrose and 2 mg/l 2,4-D. The calli were irradiated on the 6th day of the first subculture.

Calli irradiation was performed during 5 min using a LG-79 helium-neon laser with a radiation wavelength of 632.8 nm and an intensity of 10 mW at the sample level. Irradiation intensity was measured using a LM-2 device (Carl Zeiss, Germany). The calli were irradiated directly in glass test tubes (10 mm in diameter) used for their culturing, and light-intensity losses in passing through the glass did not exceed 12 %. Before and after irradiation, the calli were grown in the dark. In each independent experiment, 100 calli were used; half of them served as a control, and the other half was treated by the helium-neon laser.

The content of the compounds under investigation in the calli was determined immediately following radiation and also after 48 h. The lipid peroxidation level was assessed spectrophotometrically (Kurganova et al. 1997) as an amount of lipid peroxidation products that react with thiobarbituric acid (TBA-reactive products). To this end, 0.2 g of callus tissue was ground in a homogenizer with 9 ml of heptane:isopropanol (1:1, v/v) mixture. The homogenate thus obtained was supplemented with 0.1 ml of saturated TBA solution and centrifuged at 4000 rev/min for 10 min. The supernatant diluted with distilled water was shaken; after phase separation, the upper heptane fraction was collected. This fraction was supplemented with ethanol (1:5, v/v), and the optical density of the mixture at 532 nm was determined using an SF-46 spectrophotometer (LOMO, Russia). In all cases, the amount of secondary lipid peroxidation products in the samples was calculated from the spectrophotometric readings using molar extinction coefficient. Means from three experiments (each including nine biological replications) and their standard errors are shown in the figure. Each biological replication included from four to seven calli.

The changes in the accumulation of secondary lipid peroxidation products in wheat tissue culture induced by low-intensity laser radiation are sufficiently clear-cut (Figure 1). TBA-reactive product content in the tissue increased from 0.73 nmol/g fr wt in the control to 1.17 nmol /g fr. wt immediately after irradiation. Within 2 days after irradiation, this difference somewhat decreased, but nevertheless remained quite significant: TBA-reactive product contents in the control and irradiated samples were 1.02 and 1.12 nmol/g fr. wt, respectively. Thus, after 48 h, the content of TBA-reactive products in control samples somewhat increased as compared to that observed immediately after irradiation. Judging from the published evidence (Smirnoff 1993), this can be related to a cyclic stress-response pattern in the wheat tissue culture caused by subculturing to a new culture medium. In this case, the phases of these cycles in the control and laser-irradiated samples most likely do not coincide with each other.

The data suggest that low-intensity laser radiation can induce lipid peroxidation in wheat tissue culture. We showed that the initial response of a plant tissue to irradiation involved an increase in the content of secondary lipid peroxidation products (see Figure 1). Moreover, we showed that, as an aftereffect, laser light stimulated the morphogenetic processes in plant tissues (Salyaev et al. 2001a and b). In our opinion, this stimulation could result from the metabolic changes induced by the alterations in the contents of compounds, such as lipid peroxidation products, formed in the primary photoreactions. The changes in cells produced by the accumulation of these compounds could serve as a signal triggering not only respective defense mechanisms, but also some secondary responses including those on the transcription level (Karu 2001). This suggestion is indirectly supported by stimulation of the morphogenetic processes in the tissue cultures of wheat and wild grasses under the effect of low-intensity coherent radiation (Salyaev et al. 2001a and b).

Thus, we suggest that a general cell response induced by laser-light irradiation can be divided into two specific responses, which do not coincide in time. The first one consists of a rapid stress effect resulting in an increase in the amount of lipid peroxidation products, and the second and longer one, are the secondary reactions related to the adaptive metabolic changes and apparently accompanied by the stimulation of morphogenetic processes.

References.

• Baraboi VA. 1991. Mechanisms of stress and lipid peroxidation, Usp Sovrem Biol 111:923-931.
• Grishko VP, Grishko VT, and Glick BR. 1999. Molecular laser technology. Biotech Adv 17:341-362.
• Karu TI. 2001. Cell mechanisms of low-intensity laser therapy. Usp Sovrem Biol 121:110-120.
• Kurganova LN, Veselov AP, Goncharova TA, and Sinitsina YuV. 1997. Lipid peroxidation and antioxidant system of protection against heat shock in pea (Pisum sativum L.). Fiziol Rast (Moscow). 44:725-730 (Rus J Plant Physiol, Eng Transl).
• Kuznetsov VlV. 2001. XII Congress of the Federation of European Societies of Plant Physiologists (FESPP, 21-25 August, 2000, Budapest, Hungary). Fiziol Rast (Moscow) 48:644-648 (Rus J Plant Physiol, Eng Transl).
• Rogatkin DA and Chernyi VV. 1999. Low-intensity laser therapy: The physical point of view on the mechanism of action and application. In: Vzaimodeistvie izluchenii i polei s veshchestvom (Interaction of radiation and fields with materials) (Denisyuk YuN et al. eds). Irkutsk: SiLaP, pp. 366-378.
• Salyaev RK, Dudareva LV, Lankevich SV, and Sumtsova VM. 2001a. The effect of low-intensity coherent radiation on the processes of morphogenesis in the wheat callus culture. Dokl Akad Nauk 376:830-832.
• Salyaev RK, Dudareva LV, Lankevich SV, and Sumtsova VM. 2001b. The effect of low-intensity coherent radiation on the callus formation in the wild cereals. Dokl Akad Nauk 379:819-820.
• Skobelkin OS, ed. 1997. Primenenie nizkointensivnykh lazerov v klinicheskoi praktike (Application of Low Intensity Lasers in Clinical Practice). Moscow, Meditsyna (In Russian).
• Smirnoff N. 1993. The role of active oxygen in the response of plants to water deficit and desiccation. New Phytol 125:27-58.
  

 

Nitrate reductase activity in spring wheat at different levels of nitrate nitrogen. [p. 124-126]

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

Nitrate reductase (NR) is an enzyme well-known to plant biologists, which acts as a limiting link in the chain of nitrate reduction to ammonium and catalyzes the first stage of nitrate reduction to nitrite. Most of the studies of NR physiological and biochemical properties were published in the 1960s-80s. In particular, plant physiologists revealed the connection between NR activity and cultural plants productivity, protein synthesis in crops grain, as well as intensity of nitrogen assimilation by the plants (Deckard et al. 1973; Rautou et al. 1977; Peshkova and Khavkin 1980). Pekker and TOkarev (1984) suggest use of NR activity for diagnosing plants nitrogen nutrition. In the last few years, interest in NR has been fostered by the discovery of NR ability to reduce nitrite to nitric oxide (NO), a molecule that plays an important role in the vital processes of animals and plants (Yamasaki and Sakihama 2000; Desikan et al. 2002; Garcia-Mota and Lamathina 2003). This article sums up the results of tests on determination of NR activity during the vegetation period of spring wheat (Skala) depending on the dose of nitrogen fertilizer. The work was targeted at finding the dependence between NR activity and the degree of nitrate nitrogen availability for the plants.

Materials and Methods. The tests were performed in vegetation vessels with the capacity of 4 kg of dry soil with low content of total nitrogen (0.009 %). The mixture with nitrate source of nitrogen was used as a nutritious mineral mixture (Thomas et al. 1979). The content of mineral nitrogen introduced in the vessels varied from 0 to 800 mg / vessel. Soil moisture in the vessels was maintained at the level of 65-70 % of total moisture capacity. 10 plants grown in the vegetation house from June to August were left in each vessel.

NR activity was determined in anaerobic medium according to the method described in Mulder et al. (1959) using disks (0.05-0.1 mm) cut out from the leaves. The samples (0.3 g of fresh tissue) were placed in retorts containing the following: 0.1 M phosphate buffer with pH 7.8 (5 ml ); neutralized 0.1 M solution of malic acid (1 ml ); 5 % solution KNO3 (1 ml ) and 3 ml of distilled water. The retorts were placed in the vacuum-desiccator, with air pumped out, and kept for 1 h at 27 C. Enzyme reaction was terminated by adding 1 ml of 10 % acetic acid and 2 ml of saturated ammonium sulfate solution. In the control sample the enzyme reaction was terminated prior to incubation of the reaction mixture. NR activity was determined on the basis of the quantity of nitrites formed as a result of enzyme reaction; their content was determined in the filtrate with 0.6 % sulfanilamide in HCI and 0.6 % solution of N-(l- naphtyl) ethylenediamine hydrochloride. The color intensity was measured on the photocolorimeter (FEK-56M) at wave length of 546 nm. The samples for determination of enzyme activity were taken during the phases of two, three, four leaves, to come into ear and blooming. The upper growing and lower green leaves were subjected to analysis. The test was repeated five times; the analytical repetition is three times. Data are presented as mean ± standard deviation.

Results and Discussion. Table 3 shows that NR activity on the medium without nitrogen introduction is minimal during all the phases of plants development as compared to the variants with nitrogen introduced. The highest enzyme activity in this option was observed in the phase of two leaves, then with the plants growth and development it gradually came down, particularly in the lower leaf. Thus, at anthesis, NR activity in the upper leaf reduced by 2.6 times, in the lower leaf, by eight times compared to two-leaf phase.

Table 3. Nitrate reductase activity in spring wheat leaves in different phases of plant development depending on the level of nitrogen nutrition 9 nmol NO-2/g (fresh weight)/1 h). Note top values are for the upper leaf; bottom values are for the lower leaf.

N dose mg/vessel	Phase of plant development
	2-leaf	3-leaf	4-leaf	heading	anthesis
0	154 ± 10 100 ± 12	132 ± 10  126 ± 12	90 ± 7 51 ± 4	73 ± 3 36 ± 2	59 ± 32 0 ± 1
40	610 ± 45 700 ± 71	485 ± 35 315 ± 30	400 ± 31 306 ± 19	213 ± 18 126 ± 10	162 ± 9 98 ± 8
100	675 ± 7 745 ± 26	555 ± 45 325 ± 29	606 ± 66 340 ± 22	335 ± 22 224 ± 14	210 ± 20 139 ± 7
200	695 ± 29 800 ± 39	600 ± 20 400 ± 28	663 ± 40 383 ± 15	962 ± 51 681 ± 31	600 ± 35 350 ± 24
400	655 ± 40 655 ± 90	586 ± 59 510 ± 41	684 ± 9 600 ± 19	1,560±179 900 ± 45	1,148 ± 56 542 ± 34
800	645 ± 30 610 ± 25	510 ± 43 462 ± 40	726 ± 21 569 ± 25	1,015 ± 79 840 ± 29	1,060 ± 54 685 ± 31

In the options with nitrogen introduction regardless of the dose, enzyme activity in the upper and lower leaves either did not differ, or was slightly higher in the lower leaf than in the upper leaf. Starting from the three-leaf phase, NR activity in the variants with nitrogen dose 40 and 100 mg/vessel gradually decreased, and more so in the lower leaf. In the variant with nitrogen dose 200 mg/vessel the enzyme activity in the upper leaf remained approximately at the same level up to the blooming phase, whereas in the lower leaf it decreased. In the variants with high nitrogen content (400 and 800 mg/vessel), NR activity by the blooming phase in the upper leaf increased and in the lower leaf did not change significantly. Thus, the lowest NR activity is characteristic of the variant without nitrogen fertilizer introduction, and in the variants with low nitrogen content, NR activity by the blooming phase decreased significantly both in upper and lower leaves.

A nitrogen dose of 200 mg/vessel is a transient value between the variants with insufficient level of nitrogen in the medium and its high level. In the plants of this variant, NR activity in the upper leaf did not decrease significantly by the blooming phase, though it fell remarkably in the lower leaf. Apparently, in this case regulation of nitrogen nutrition by the plant is directed primarily at providing growing organs with mineral nitrogen, which causes the flow of newly absorbed nitrates to go to the growing leaf ensuring fairly high enzyme activity.

In the upper and lower leaves at low nitrogen doses (0, 40, and 100 mg) there were observed identical regularities of NR activity change: by the blooming phase NR activity in the upper leaf decreased in accordance to the doses by 2.6; 3.8, and 3.2 times, and in the lower leaf by 8.0; 7.1, and 5.4 times. In the variant with nitrogen dose of 200 mg activity, only insignificantly decreased in the upper leaf by the blooming phase (approximately by 1.2 times), in the lower leaf the enzyme activity decreased by 2.3 times. At high nitrogen availability (400 and 800 mg/vessel), NR activity was, as a rule, high in both upper and lower leaves.

The lack of nitrogen results in a decrease of NR activity first in the lower leaf, whereas in the upper leaf, enzyme activity remains higher. This effect is well seen particularly in the variant with nitrogen dose of 200 mg/vessel. Attention should be paid to the intensification of NR activity during spike formation in the variants with high nitrogen availability for the plants, which, apparently, may be accounted for by intense nitrates absorption by the plants during this vegetation period, as formation of reproduction organ creates a powerful attracting center and new growth points, which require inflow of nitrous substances for synthetic processes.

Sufficient nitrogen availability allows the plant to maintain NR activity at a high level both in the upper and lower leaves. This, apparently, leads to prolonging of the lower leaves life via activation of nitrogen assimilation processes, and, consequently, to a slowdown of the plant ageing process and a prolonging of the wheat plant's vegetation period.

Thus, the acquired data speak in favor of the connection between NR activity in the leaves and the level of mineral nitrogen availability for the plants. These results are of interest for elaboration and specification of methods of diagnostics of grain-crops nitrogen nutrition using NR activity. In particular, according to our data in the course of diagnostics of nitrogen availability for spring wheat by NR activity, it is efficient to determine enzyme activity not only in the upper growing leaf, but also in the lower green leaf, which has completed growth, for the sake of earlier identification of soil nitrogen identification.

References.

• Dalling MJ and Loyn RH. 1977. Level of activity of nitrate reductase at the seedlings stage as predictor of grain nitrogen yield in wheat (Triticum aestivum L.). Aus J Agric Res 28:1-11.
• Deckard EZ, Lambert RJ, and Hageman RH. 1973. Nitrate reductase activity in corn leaves as related to yields of grain and grain protein. Crop Sci 13:343-351.
• Desikan R, Griffiths R, Hancock J, and Neil S. 2002. A new role for on old enzyme: nitrate reductase - mediated nitric oxide generation is required for abscisic acid induced stomatal closure in Arabidopsis thaliana. Proc Natl Acad Sci USA 99:16314-16318.
• Garcia-Mata C and Lamathina L. 2003. Abscisic acid, nitric oxide and stomatal closure - is nitrate reductase one of missing links? TRENDS Plant Sci 8:20-26.
• Mulder EG, Boxma R, and Van Veen WL. 1959. The effect of molybdenum and nitrogen deficiencies on nitrate reduction in plant tissues. Plant and Soil 10:335-345.
• Pekker EG and Tokarev BI. 1984. The biochemical approach to diagnostics of security of plants by nitrogen in a course of vegetative period. In: Nitrogen exchange and crops productivity in the chimization of agriculture in Western Siberia (Tokarev BI, Ed). Novosibirsk, pp. 27-42 (In Russian ).
• Peshkova AA and Khavkin EE. 1980. Nitrate reductase activity and nitrates assimilation in connection with the growth speed of maize sprouts. Fiziologia rastenii 27:1032-1039 (In Russian).
• Rautou S, Boyat A, and Robin P. 1977. Because of the strategic role of nitrate reductase in nitrogen metabolism, relationschips between activity and productivity or quality of the kernel have been the subject of much research. Maize Genet Coop Newslet 51:27-29.
• Thomas RJ, Feller H, and Erismann KN. 1979. The effect of different inorganic nitrogen sources and plant age on the composition of bleeding sap of Phaseoules vulgaris. New Phytol 82:657-669.
• Yamasaki H and Sakihama Y. 2000. Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Lett 468:89-92.

 

Impact of disulfidreductase from the wheat caryopsis on certain technological characteristics of wheat flour and dough. [p. 126-127]

S.V. Osipova, A.V. Permyakov, and T.N. Mitrofanova, and T.N. Pshenichnikova, M.F. Ermakova, and
A.K. Chistyakova (Institute of Genetics and Cytology, Siberian Russian Academy of Sciences, Lavrentyev Ave. 10, Novosibirsk).

The technological quality of wheat flour is known to be largely dependent on highly-molecular subunits of glutenin containing the biggest amount of SH-groups, capable of forming S-S links ( Payne 1987). Dependence of gluten quality on the content of disulfide links was experimentally proven by Kretovich et al. (1978), Vakar et al. (1972 ), Bloksma (1975), and Kaczkowski et al. (1980). In our laboratory, we extracted the enzyme with thiol:proteindisulfide oxidoreductase (EC 1.8.4.2) activity from wheat corn (Trufanov et al. 1999). The enzyme catalyzes dissociation of protein disulfides using restored glutathione as a cofactor. Our model experiment has shown that wheat disulfidreductase reduces aggregation ability of gluten proteins, which is apparently caused by dissociation of proteins SS-links and consequent weakening of gluten matrix rigidity (Osipova et al. 2004). The investigation was focused on the impact of disulfidreductase preparation introduction in the course of dough kneading on certain technological characteristics of flour and dough.

Materials and Methods. Disulfidreductase was extracted and purified from flour of Siberian selection wheat, Tulunskaya 12 variety as per the earlier described methods (Trufanov et al. 1999). In the tests there were used partially purified disulfidreductase preparation containing admixture of glutathionreductase (N1) and preparation of disulfidreductase with enzyme purity, that is not containing admixtures of other thiol-disulfide metabolism (N2) enzymes. Fifty mg of enzyme preparation containing approximately 3-5 units of disulfidreductase activity and NaCl were added to 50 g of flour of cultivar Mironovskaya 808. A similar portion (50 mg) of NaCl was added to control. Technical parameters were analyzed in compliance with the methods of the State Variety Testing of Agricultures (Methods 1988). The physical properties of dough were studied using Chopin alveograph, which was used to determine specific work for dough deformation (strength of flour, W; units of alveograph, u.a.), tenacity (P, mm) and extensibility (L, mm), and P/L ratio. Table 4 presents average data of three independent tests ± standard error.

Results and Discussion. The addition of enzymes only insignificantly increases dough tenacity (P) (Table 4). Dough extensibility (L) values increase remarkably, N2 preparation addition increases extensibility by 7 %, whereas the addition of partially purified preparation (N1) by approximately 17 %. High efficiency of N1 preparation may be accounted for by the fact that presence of glutathionreductase ensures fairly high amount of restored glutation required for catalytic activity of disulfidreductase. Flour strength reliably increases with the addition of enzyme preparations. P/L proportion, however, practically does not change, so on the whole gluten quality of the Mironovskaya 808 wheat does not improve. Nevertheless, significant, up to 17 %, increase of dough extensibility is of interest. These data allow us to infer that partially cleaned disulfidreductase preparation containing glutathionreductase activity may be used to improve qualities of excessively strong, short-tearing gluten.

Table 4. Alveograph parameters with enzyme additives introduction (Significant at * P > 0.05; ** P > 0.01).

Sample	Alveograph parameters
	Flour strength, W, u.a.	Tenacity, P, mm	Extensibility, L, mm	P/L ratio
Control	188 ± 8	60 ± 0.5	112 ± 9	0.5 ± 0.04
Treatment
Preparation N1	223 ± 6**	64 ± 4.0	131 ± 10*	0.5 ± 0.10
Preparation N2	231 ± 2**	67 ± 4.0	120 ± 15	0.6 ± 0.10

References.

• Bloksma AH. 1975. Thiol and disulfide groups in dough rheology. Ceeal Chem 52:170-183.
• Gosagroprom M. 1988. Methods of state variety testing of agricultures.
• Kaczkovski J and Meleszko T. 1980. The role disulfide bonds and their localization in wheat protein molecules. Ann Tech Agric 29:377-384.
• Kretovich VL and Zhmakina OA. 1978. On the nature of gluten links. Rep AS USSR 238:985-987.
• Osipova SV, Permyakov AV, Mitrofanova TN, and Trufanov VA. 2004. Thiol:protein disulfide oxidoreductase (EC 1.8.4.2) in wheat caryopses. C. Impact on aggregation of wheat storage proteins. Ann Wheat Newlet 50:144-145.
• Payne PI. 1987. Genetics of wheat storage proteins and the effect of allelic variation on breadmaking quality. Ann Rev Plant Physiol 38:141-153.
• Trufanov VA, Kichatinova SV, and Permyakov AV. 1999. Enzymes of the thiol-disulfide exchange in wheat and their effect on gluten proteins. Prikl Biochem Microbiol 35:219-222.
• Vakar AB, Demidov VS, and Zabrodina GM. 1972. Investigation of physical-chemical differences of various quality gluten. Appl Biochem Microbiol 8:292-302.

 

Isoforms of glutathione reductase in wheat grains Triticum aestivum. [p. 127-128]

A.V. Permyakov, T.N. Mitrofanova, and S.V. Osipova.

Glutathione reductase (GR) catalyzes the NADPH-dependent reduction of oxidized glutathione (Foyer et al. 1997). The primary function of glutathione reductase is to maintain a high GSH/GSSG ratio in cells, which is crucial for a variety of cellular functions, including the biosynthesis of DNA (Noctor et al. 1998). This flavoprotein oxidoreductase has a central role in maintaining GSH within the cellular environment, particularly during stress. Most, if not all, stresses include an oxidative stress component (Prasad et al. 1994; Wise 1995) that leads to tissue damage if antioxidative defenses are insufficient.

Wheat leaves contain two charge/mass-separable isoforms of glutathione reductase, one chloroplastic and the other probably cytosolic (De Lamotte et al. 2000). The endosperm of durum mature kernels contained a single form of glutathione reductase; it appeared about the 18th day after anthesis, whereas another isoform, present at the early stages of grain development, disappeared between the 20th and 30th days after flowering (Lascano et al. 2001).

Materials and Methods. Grains of the wheat cultivar Tulunskaya-12 were used in this research. The soluble, enzymatically active protein fraction of the wheat grains was extracted with a 0.1 Tris buffer, pH 7.5, containing 2 mM EDTA, from standard ground flour in the proportion 1:2 (weight:volume). The extract was subjected to chromatography on DEAE-Sephadex A-50. The protein fraction was eluted in a 0-0.6 M NaCl gradient. Fractions obtained were separated according in 7 % PAAG to the method of Davis (1964) at the basic pH. Molecular forms of glutathione reductase were identified immediately on gel slabs by specific coloring (Ye et al. 1997). Bromophenol-blue was used as the marker for estimating Rf values, indicating the mobility of enzyme bands relative to the mobility of the bromophenol-blue front.

Results and Discussion. Selective enzyme coloring after native electrophoresis in the PAAG slabs manifested its presence in all the protein fractions acquired through ion-exchange chromatography of DEAE-sephadex A-50 (Figure 2), revealing that the enzyme is represented by two molecular forms with different relative electrophoretic mobility (Rf) 0.36 and 0.32 (GR 1 and GR 2, respectively), which split during proteins desorption from anions by a linear gradient of NaCL concentration. The initial enzyme extract contained both enzyme isoforms (Figure 2, lane 1). During ion-exchange chromatography, the first to eluate from the sorbent was glutathione reductase molecular form (GR 2) with Rf 0.32 (Figure 2, lanes 25-32), then GR 1, with Rf 0.36 (Figure 2, lanes 33 and 34). Thus, unlike the grain of complete maturity T. turgidum subsp. durum (Lascano et al. 2001), T. aestivum subsp. aestivum grains contain two isoforms of glutathione reductase that may be split with the help of ion-exchange chromatography.

References.

• Davis BJ. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 121:404-427.
• De Lamotte F, Vianey-Liaud N, Duviau MP, and Kobrehel K. 2000. Glutathione reductase in wheat grain. 1. Isolation and characterization. J Agric Food Chem 48:4978-4983.
• Foyer CH, Lopez-Delgado H, Dat JF, and Scott IM. 1997. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signaling. Physiol Plant 100:241-254.
• Lascano HR, Casano LM, Melchiorre MN, and Trippi VS. 2001. Biochemical and molecular characterization of wheat chloroplastic glutathione reductase. Biologia Plant 44:509-516.
• Noctor G and Foyer C. 1998. Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249-279.
• Wise RR. 1995. Chilling-enhanced photooxidation: the production, action and study of reactive oxygen species produced during chilling in the light. Photosynth Res 45:79-97.
• Ye B, Gitler C, and Gressel J. 1997. A high-sensitivity, single-gel, polyacrylamide gel electrophoresis method for the quantitative determination of glutathione reductases. Anal Biochem 246:159-165.

 

The oxidative phosphorylation uncoupling of winter wheat mitochondria by saturated fatty acid and participation of ADP/ATP-antiporter. [p. 128-130]

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

Oxidative phosphorylation is the main ATP source in aerobic organisms. In certain conditions, the uncoupling of oxidative phosphorylation (substrate oxidation without phosphorylation) occurs. Fatty acids are natural uncouplers of oxidative phosphorylation. Different mechanisms through which fatty acids cause uncoupling are known (Jezek 1999; Skulachev 1999; Wieckowski et al. 2000; Kadenbach 2003). Recent studies indicates that fatty acids are also involved in cell death pathways (Penzo et al. 2002). Fatty acid uncoupling is inhibited by addition of bovine serum albumin (BSA), purine nucleotides, and by ATP/ADP antiporter inhibitor, carboxyatractyloside (CAT) (Skulachev 1991).

As shown previously, unsaturated free fatty acids in winter wheat mitochondria plays not only a role of uncouplers (oleic, petrozelinic, linoleic and erucic acids), but also could be the only oxidation substrate for them (linoleic acid) (Grabelnych et al. 2004). Although unsaturated fatty acids have more effect on the mitochondrial membrane potential, the role of saturated fatty acid in energetic cell metabolism is significant too (Pastore et al. 2000; Penzo et al. 2002). Lauric and palmitic acids have caused more significant y decrease rate in durum wheat mitochondria among the saturated acids (Pastore et al. 2000). Fatty acid-dependent uncoupling of oxidative phosphorylation plays an adaptive role during hypothermia and oxidative stress in the plant mitochondria (Casolo et al. 2000; Pastore et al. 2000).

The aim of the present investigation is to study the influence of saturated fatty acid on the winter wheat mitochondria function and determine the ADP/ATP participation in fatty acid-dependent uncoupling.

Materials and Methods. Three-day-old etiolated shoots of winter wheat cultivar Zalarinka germinated on moist paper at 26 C, were used in this work. Mitochondria were extracted from winter wheat shoots by differential centrifugation as describes previously (Pobezhimova et al. 2001). Isolated mitochondria were resuspended in the following medium: 40 mM MOPS-KOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2.

Mitochondrial activity was recorded polarographically at 27 C using a platinum electrode of a closed type in a 1.4 ml volume cell (Estabrook 1967). The reaction mixture contained 125 mM KCl, 18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. 10 mM Malate in the presence of 10 mM glutamate was used as an oxidation substrate. Lauric (C 12:0), palmitic (C 16:0), stearic (C 18:0), and behenic (C 22:0) acids were used in concentrations from 0,056 mkM to 10 mM. 1 mkM carboxyatractyloside (Catr) was used as ADP/ATP inhibitor.

Polarograms were used to calculate the rates of phosphorylative respiration (state 3), nonphosphorylative respiration (state 4), the rate of respiration after fatty acid addition, the rate of respiration after Catr addition, respiration control by Chance-Williams, and the ADP:O ratio (Estabrook 1967). The concentrations of mitochondrial protein and CSP 310 were analyzed by Lowry method (Lowry et al. 1951). All the experiments were made in three-six preparations. The data obtained were analyzed statistically, i.e., arithmetic means and standard errors were determined.

Results and Discussion. To determine saturated fatty acid uncoupling effect we added different amounts of fatty acid to state 4 mitochondria, because fatty acid-dependent increase of state 4 oxygen consumption is one of the main indicators of fatty acid-dependent uncoupling together with coefficient RC and ADP:O ratio decrease.

We found that an addition to state-4 mitochondria physiological concentrations (0.056 and 0.15 mkM) of lauric and palmitic acids didn't cause any changes in the rate of oxygen consumption but addition physiological concentrations of stearic and behenic acids caused even a decrease of oxygen consumption (Figure 3). Behenic acid did not cause any changes in the rate of oxygen consumption at higher concentrations added. The most influence of lauric acid on the rate of oxygen consumption the was found at concentration 50 mkM, which caused about 54 % stimulation of non-phosphorylative respiration (Figure 3). Palmitic acid also has the most uncoupling activity at concentration 50 mkM, which cause twofold increase of non-phosphorylative respiration. The uncoupling effect of stearic acid become apparent only at high concentrations and didn't exceed 30 % of stimulation of state 4 respiration at concentration 500 mkM (Figure 3). Recoupled effect of Catr (about 39 % decrease of non-phosphorylation respiration rate) in winter wheat mitochondria shows participation of ADP/ATP-antiporter in this process. The uncoupling effect of palmitic and stearic acid was fully associated with participation of ADP/ATP-antiporter. The uncoupling effect of lauric acid also was partially associated with participation of ADP/ATP-antiporter.

The other picture of respiration stimulation in fatty acid-treated mitochondria after ADP addition was observed. In second cycle of phosphorylation the uncoupling action of all studied saturated fatty acids, including behenic acid, was found. Figure 4 shows the data about fatty acid influence on the oxygen consumption at concentrations which cause maximal uncoupling in the second cycle of phosphorylation. Fatty acids can be divided into two groups: 1) lauric and palmitic acid (Figure 4A and 4B) that cause immediate stimulation of state-4 respiration and the same or smaller stimulation of state-4 respiration in second cycle of phosphorylation; 2) stearic and behenic acids (Fig\ure 4C and 4D), that did not cause stimulation of state-4 respiration immediately but cause stimulation of state-4 respiration in second cycle of phosphorylation.

An addition of 50 mkM lauric acid caused the increase in state-4 respiration (Figure 4A, 3), the subsequent addition of ADP caused the transfer of mitochondria to phosphorylative state (Figure 4A, 4) and then to nonphosphorylative state (Figure 4A, 5), whose value was equal to respiration rate in first cycle of phosphorylation after addition lauric acid. Palmitic acid also caused the increase in state-4 respiration (Figure 4B, 3) and subsequent addition of ADP accompanied transfer to state 3 (Figure 4B, 4), but its value was smaller than in first cycle of phosphorylation. State-4 respiration was higher (Figure 4B, 5) as compared with state-4 respiration before palmitic acid addition, but smaller then state-4 respiration after palmitic acid addition in first cycle of phosphorylation (Figure 4B, 2).

Although 5 mkM stearic acid addition in state 4 did not cause statistically significant stimulation of respiration (Figure 4C, 3), we observed 58 % increase of non-phosphorylative rate of respiration in second cycle of phosphorylation (Figure 4C, 4). Likewise the influence of stearic acid, behenic acid addition in state 4 didn't cause stimulation of respiration (Figure 4D, 2), but we observed 56 % increase of non-phosphorylative rate in the second cycle of phosphorylation (Figure 4D, 4). In all cases the increase of non-phosphorylative respiration rate in second cycle of phosphorylation was accompanied by decrease of RC coefficient and ADP:O ratio (Table 5). RC coefficient decrease was 26-49 % and ADP:O ration decrease was 27-33 %. So, all saturated fatty acids studied cause the uncoupling of oxidative phosphorylation in second cycle phosphorylation; the increase of non-phosphorylation respiration and decrease of RC coefficient and ADP:O ratio.

Table 5. The decrease of RC coefficient and ADP:O ratio in winter wheat mitochondria in presence of fatty acids during second cycle of phosphorylation. 100 % is RC coefficient or ADP:O ratio of mitochondria at first cycle of phosphorylation before fatty acid addition.

Saturated acid concentration	RC coefficient (%)	ADP:O ratio (%)
Lauric, 50 mkM	66.16	72.47
Palmitic, 10 mkM	51.00	71.27
Stearic, 5 mkM	58.01	67.21
Behenic, 5 mkM	73.87	66.86

The stimulation of respiration by lauric acid was fully associated with ADP/ATP-antiporter participation (Figure 4A, 6). At the same time, the stimulation of respiration by palmitic, stearic and behenic acids decreased after addition of Catr only about 50 % (Figure 4B, C, D, 6). Therefore, we conclude that different carrier proteins participate in these fatty acid-dependent uncoupling.

All saturated fatty acids studied were able to cause the increase of winter wheat mitochondria respiration. Lauric, palmitic, and stearic acids caused oxidative phosphorylation uncoupling immediately, at the same time the uncoupling effect of behenic acid started after subsequent ADP stimulation. In our experiments with T. aestuvum subsp. aestivum, it is shown that palmitic acid have the most uncoupling effect, whereas Pastore with coauthors (2000) found using T. turgidum subsp. durum mitochondria that lauric acid have the most uncoupling effect. ADP/ATP-antiporter participated in fatty acid-dependent uncoupling the all studied saturated acids.

Acknowledgments. The work has been performed, in part, with the support of the Russian Science Support Foundation, Russian Foundation of Basic Research (projects 03-04-48151 and 05-04-97231) and Siberian Division of Russian Academy of Sciences Youth Grant (project 78).

References.

• Casolo V, Bradiot E, Chiandussi E, Macri F, and Vianello A. 2000. The role of mild uncoupling and non-coupled respiration in the regulation of hydrogen peroxide generation by plant mitochondria. FEBS Lett 474:53-57.
• Estabrook RW. 1967. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratio. Methods Enzymol 10:41-47.
• Grabelnych OI, Pivovarova NYu, Pobezhimova TP, Kolesnichenko AV, Sumina ON, and Voinikov VK. 2004. The influence of monounsaturated acid fatty acid on the function of winter wheat mitochondria. Ann Wheat Newslet 50:128-131.
• Jezek P. 1999. Fatty acid interaction with mitochondrial uncoupling proteins. J Bioenerg Biomembr 31:457-466.
• Kadenbach B. 2003. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim Biophys Acta 1604:77-94.
• Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with Folin phenol reagent. J Biol Chem 193:265-275.
• Pastore D, Fratianni A, Di Pede S, and Passarela S. 2000. Effects of fatty acids, nucleotides and reactive oxygen species on durum wheat mitochondria. FEBS Lett 471:88-92.
• Penzo D, Tagliapietra C, Colonna R, Petronilli V, and Bernardi P. 2002. Effects of fatty acids on mitochondria: implications for cell death. Biochim Biophys Acta 1555:160-165.
• Pobezhimova TP, Grabelnykh OI, Kolesnichenko AV, Sumina ON, and Voinikov VK. 2001. Localization of proteins immunologically related to subunits of stress 310-kD protein in winter wheat mitochondria. Russ J Plant Physiol 48:204-209.
• Skulachev VP. 1991. Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation. FEBS Lett 294:158-162.
• Skulachev VP. 1999. Anion carriers in fatty acid-mediated physiological uncoupling. J Bioenerg Biomembr 31:431-445.
• Wieckowski MR, Brdiczka D, and Wojtczak L. 2000. Long-chain fatty acid promote opening of the reconstituted mitochondrial permeability transition pore. FEBS Lett 484:61-64.

 

Seasonal changes in the content of dehydrin-like proteins in mitochondria from crowns of field-grown winter wheat plants. [p. 132-133]

I.V. Stupnikova, G.B. Borovskii, A.I. Antipina, and V.K. Voinikov.

Cold stress adaptation has been established to induce dehydrin synthesis and accumulation (Close 1996). These proteins belonging to the family of the late embryogenesis proteins are characterized by the presence of highly conserved Y, S, and K segments. A peculiarity of dehydrin amino-acid composition provides for their functions. Amphiphilic-helices found in dehydrins are considered to bind to intracellular molecules, especially membranes and proteins, mainly by hydrophobic interactions (Close 1997). These associations may protect the functions of membranes and proteins by preventing coagulation during environmental stress and maintain water in dehydrated cells (Hoekstra et al. 2001). A correlation was observed between the endogenous concentration of dehydrins and frost tolerance of plant species, organs, or tissues (Sarhan et al. 1997). The patterns of dehydrin accumulation in trees coincided with seasonal fluctuations in their frost resistance (Wisniewski et al. 1996). In tissues of leaves (deciduous species), flower buds, blueberry bark, peach, black current, poplar, willow, and acacia, the accumulation of dehydrin-like proteins was reported in autumn and winter. However, the seasonal changes in the composition and content of these proteins in wintering grasses have not been studied. The presence of dehydrins in the nucleus, cytoplasm, cytoskeletal elements, and plasma membrane of various cells in seedlings and adult plants was demonstrated by immunolocalization and subcellular fractionation (Houde et al. 1995). Earlier, we detected two dehydrin-like proteins, which accumulated in mitochondria of the freezing-resistant wheat seedlings during cold hardening under laboratory conditions (Borovskii et al. 2000). Taking into account all facts above the objective of the study was to follow possible seasonal fluctuations in amount and spectra of dehydrins in winter wheat crowns being the vitally important winter wheat part, which determines winter survival.

Materials and Methods. Experiments were performed on field-grown winter wheat plants cultivar Irkutskaya ozimaya hardened under natural conditions. Mitochondria were isolated by differential centrifugation, as was described in (Borovskii et al. 2000). The crowns were ground with a mortar and pestle in the medium containing 0.3 M sucrose, 40 mM Mops (Sigma, United States), 2 mM EDTA, 10 mM KCl, 1 mM MgCl2, 0.05 % polyvinylpyrrolidon, 0.5 % cysteine, and 0.5 % BSA (ICN, United States), pH 7.5. All other procedures were performed similarly as during mitochondria isolation from seedlings. Mitochondria from the crowns were purified using a discontinuous Percoll gradient comprising 21 % and 35 % Percoll. Purified mitochondria were used for protein isolation. SDS-PAGE was run in 11 % acrylamide gel in the modified Laemmli system (Laemmli 1970). Immunodetection was performed by the method of Timmons and Dunbar (1990). Polyclonal antibody against K fragment of dehydrins was kindly presented by T.J. Close (University of California, Riverside).

Results and Discussion. All our previous experiments concerning the detection of mitochondrial dehydrins were carried out with seedlings. Therefore, elucidating whether or not similar accumulation of dehydrins occurred in mitochondria of overwintering adult plants was important. Mitochondria were isolated from winter wheat crowns during autumnal hardening, in winter, and during spring deadaptation. The mitochondria from crowns contained proteins with molecular weights being similar to proteins from seedlings, 63 and 52 kD (Figure 5). Any other proteins related to dehydrins were not detected. The content of these proteins increased substantially during autumnal hardening. Peshkova et al. (1998) showed that the crowns of this winter wheat cultivar lost up to 67-69 % of water during hardening. The highest content of mitochondrial dehydrins was in December, and thereafter it began to decline gradually. In spring, the content of dehydrins in mitochondria from wheat crowns decreased to their level in autumn. In April, dehydrin content was similar to that in September; in May, it was even lower (Figure 5). We know that winter wheat cryotolerance increases during cold acclimation starting from early autumn and achieves its highest value by the beginning of winter, indicating the interrelation between dehydrin content and winter wheat freezing tolerance, what is in agreement with the results of other researchers (Sarhan et al. 1997). Earlier we showed that in contrast to mitochondrial dehydrins, the spectra of those in total cell water-soluble fraction contained much more protein groups. In the total water-soluble protein fraction, both the amount of dehydrins in each group and the number of dehydrin groups of a particular molecular weight increased with autumnal hardening and attained the highest values by the end of January. At this time, dehydrins with molecular weights of 209, 196, 66, 50, 41, 24, 22, 17, 15, and 12 kD were detected among soluble proteins (Stupnikova et al. 2004). Two protein groups (66 and 50 kD) had molecular weights close to those of mitochondrial dehydrins (63 and 52 kD). The methods used did not permit us to decide whether these proteins with close molecular weights were similar or different proteins.

Adaptation process is known to include two stages: unspecific stress-response and specific adaptation (Kuznetsov et al. 1987). During the first stage, mobilization and development of plant defensive systems occur, which provide for organism short-term surviving. During the second stage, specific adaptation mechanisms develop, which are responsible for plant life under the conditions of long-term stress (Kuznetsov et al. 1987). The accumulation of these dehydrins during cold adaptation of field-grown plants and the absence of their further changes in autumn and winter indicate evidently that these proteins operate like cryoprotectors during both stages of adaptation.

References.

• Borovskii GB, Stupnikova IV, Antipina AI, Downs CA, and Voinikov VK. 2000. Accumulation of dehydrin-like proteins in the mitochondria of cold-treated plants. J Plant Physiol 156:797-800.
• Close TJ. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97:795-803.
• Close TJ. 1997. Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol Plant 100:291-296.
• Hoekstra FA, Golovina EA, Buitink J. 2001. Mechanisms of plant desiccation tolerance. TRENDS Plant Sci 6:431-438.
• Houde M, Daniel C, Lachapelle M, Allard F, Laliberte S, and Sarhan F. 1995. Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. Plant J 8:583-593.
• Kuznetsov VV, Kimpel J, Gokdzhiyan D, and Ki J. 1987. Elements of nonspecificity of the plant genome responses to cold and heat stresses. Fiziol Rast (Moscow) 34:859-868 (Sov Plant Physiol, Eng Transl).
• Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227:680-685.
• Peshkova AA and Dorofeev NV. 1998. Winter tolerance of winter wheat according to conditions of vegetative growth and mineral nutrition. Agrokhimiya 6:26-33.
• Sarhan F, Ouellet F, and Vazquez-Tello A. 1997. The wheat WCS120 gene family: a useful model to understand the molecular genetics of freezing tolerance in cereals. Physiol Plant 101:439-445.
• Stupnikova IV, Borovskii GB, and Voinikov VK. 2004. Seasonal changes in the composition and content of dehydrins in winter wheat plants. Rus J Plant Physiol 51:1-7.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.
  Wisniewski M, Close TJ, Artlip T, and Arora R. 1996. Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants. Physiol Plant 96:496-505.

 

An effect of varied stresses on alternative oxidase and plant uncoupling mitochondrial protein expression in winter wheat seedlings. [p. 133-135]

A.A. Truhin, E.L. Tauson, and G.B. Borovskii.

The different stress effectors are able to create similar damages in cell: the increased viscidity of membranes, formation of gel phases, degradation, and destruction of proteins, phospholypids and other macromolecules. An identical phenomenon was shown during treatment of cells by superoxide (Vanlerberghe et al. 1992). Accumulation of active oxygen and its derivatives also have been shown in plants in response to various environmental stresses. Oxidizing stress can be considered as secondary answer to damage of cell organelles and activation of redox-enzyme system in the cell (Vanlerberghe et al. 1997). Recent reports show the existence of various pathways of this process leading to similar results - decreasing of transmembrane potential on inner membrane of mitochondria and reduction of mitochondrial reactive oxygen species (ROS) generation. Protein participation in this type of regulation depends on plant species, stage of the development, type of stress, and some other factors. Alternative oxidase (AOX) and plant uncoupling mitochondrial proteins (PUMP) seems to play a significant role in ROS production regulation processes. These proteins decrease transmembrane potential in different ways. Activation of the AOX pathway leads to uncoupling of oxidation and phosphorylation in three of four respiratory complexes. On the other hand, PUMP (like other members of UCP (uncoupling protein) family) does not block phosphorylation, but stimulates the spreading of potential via transport of fatty acids ions (Umbach 1993). The correlation of these two ways in decreasing of transmembrane potential is of great interest. We note that AOX and PUMP can work as antagonists because fatty acids that necessary for PUMP functioning depresses AOX activity (Jezek et al. 2000). The aim of the present study was to demonstrate the amounts of AOX and PUMP expression patterns in winter wheat under different stressing conditions using multiplex RT-PCR method.

Materials and methods. After growing for 3 days, wheat seedlings were subjected to environmental stress treatments. Low temperature heat and dehydration treatments were conducted by transferring of etiolated seedlings to separate conditions: 4 or 39 C, 30 % H2O2 solution for 1.5 h, polyethylene glycol (PEG MW8000) solution treatment for 3 h, and dehydration in air for 24 and 48 h. Samples were immediately frozen in liquid nitrogen and stored at -70 C until processed for RNA extraction. Transcript levels of aox and pump was also controlled under unstressed conditions.

Primers were designed from published sequence data of genes encoding AOX, PUMP (EMBL database) and RDF (Monstein et al. 2002) to amplify 415-bp, 505-bp, and 890-bp fragments of corresponding transcripts from T. aestivum cv. Irkutskaya ozimaya. Using a SV Total Isolation System (Promega, Madison, WI), 200 ng of total RNA was isolated and used for the first-strand cDNA synthesis with REVERTA kit (Amplisense, Moscow) and 3'-primer. The amount of total RNA in reactions was identical in experiments which results we compared. The transcript levels of the constitutively expressed rdf gene were also monitored on corresponding gels to confirm that amount of RNA in compared experiments was equal.

Results and Discussion. The early events of plant adaptation to environmental stresses are perception and subsequent stress-signal transduction that lead to the activation of various physiological and metabolic responses, including the development of oxidation stress. Therefore, they can cause induction of aox and pump genes expression since AOX and PUMP have been shown efficiently reduce the generation of mitochondrial reactive oxygen species (Kowaltowski et al. 1998; Umbach 1993).

The expression patterns of aox and pump were analyzed under various stresses. Expression of aox and pump were not included under high temperature treatment (Figure 6) or short-time cold stress (Figure 7), but aox expression was induced after 24 hour of low temperature treatment whereas pump gene expression was not affected (Figure 7). Expression of aox was induced after 48 h of dehydration (Figure 8). The strongest and fastest accumulation of aox and pump mRNAs within 1,5 h was observed when wheat seedlings were subjected to high oxidative stress with hydrogen peroxide (Figure 9).

These results demonstrate that expression of pump was induced in winter wheat seedlings mainly by strong oxidative stress. Previously, Maia et al. (1998) reported that cold-inducible AtPUMP protein may play a role in heat-requiring physiological events in Arabidopsis. Although, two cDNAs encoding UCP-like proteins have been isolated from potato (StUCP) (Laloi et al. 1997). Because the expression of StUCP was detected mainly in flowers and fruits, it has been hypothesized that StUCP can be associated with burst of respiration in flowering and fruit ripening in combination with AOX (Laloi et al. 1997). The cold treatment at 4 C for 12 h of skunk cabbage plants (Symplocarpus foetidus) was enough for expression of two UCP-like genes SfUCP and SfUCP encoding uncoupling proteins (Ito 1999).

Only powerful oxidative stress with hydrogen peroxide could induce strong and simultaneous expression of aox and pump genes in winter wheat seedlings (Figure 9). We propose from our results that aox induction most likely and mainly occurs as part of the acclimation process and is necessary for adaptation to stress conditions. aox, therefore, is a gene of adaptation. This possibility is supported by other investigators (Djajanegara et al. 2002). On the other hand, it may be that two energy-dissipating systems, both PUMP and AOX are involved in the regulation of the oxidative stress development in extreme situation of oxidative burst as it was in our experiment with hydrogen peroxide treatment.

References.

• Djajanegara I, Finnegan P, Mathieu C, McCabe M, and Whelan J. 2002. Regulation of alternative oxidase gene expression in soybean. Plant Mol Biol 50:735-742.
• Ito K. 1999. Isolation of two distinct cold inducible cDNAse encoding plant uncoupling proteins from the spadix of skunk cabbage (Simplocarpus foetidus) Plant Sci 149:167-173.
• Jezek P, Borecky J, Zackova M, Costa A, and Arruda P. 2000. Possible basic and specific functions of plant uncoupling proteins (pUCP). Bioscience Rep 21:183-189.
• Kowaltowski AJ, Costa AT, and Vercesi E. 1998. Activation of the potato plant uncoupling mitochondrial protein inhibits reactive oxygen species generation by the respiratory chain. FEBS Lett 425:213-216.
• Laloi M, Klein M, Riemeier JW, Muller-Rober B, Fleury C, Bouillaud F, and Ricquier D. 1997. A plant cold-induced uncoupling protein. Nature 389:135-136.
• Monstein H and Ellnebo-Svedlund R. 2002. Molecular typing of Helicobacter pylori by virulence-gene based multiplex PCR and RT-PCR analysis. 7:182-189.
• Umbach A and Siedow N. 1993. Covalent and noncovalent dimers of the cyanide-resistant alternative oxidase protein in higher plant mitochondria and their relationship to enzymes activity. Plant Physiol 103:845-854.
• Vallone M, Just S, Coble D, and Butler M. 2004. A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int J Legal Med 118:147-157.
• Vanlerberghe GC and McIntosh L. 1992. Lower grows temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol 100:115-119.
• Vanlerberghe GC and McIntosh L. 1997. Alternative oxidase: from gene to function. Ann Rev Plant Physiol Plant Mol Biol 48:703-734.
  

 

Dehydrin localization in mitochondria of winter wheat seedlings. [p. 135-137]

I.V. Stupnikova, G.B. Borovskii, A.I. Antipina, and V.K. Voinikov.

Among proteins related to cold acclimation and cold stress tolerance, the dehydrin family presents peculiar interest. This family, known as a group-2 late embryogenesis abundant (LEA) proteins, is one of the ubiquitous water-stress-responsive proteins in plants (Close 1997). These proteins are hydrophilic, thermostable and glycine-rich and possess unique repeated sequences that are believed to form putative amphiphilic a-helices. The putative multiple functions of dehydrins have been described from the viewpoint of deduced secondary structures. They were hypothesized to function by stabilizing large-scale hydrophobic interactions such as membrane structures or hydrophobic patches of proteins. Highly-conserved polar regions of dehydrins have been suggested to hydrogen-bond with polar regions of macromolecules, acting essentially as a surfactant, to prevent coagulation under conditions of cellular dehydration or low temperatures (Ismail et al. 1999). Immunolocalization and subcellular fractionation results have showed that members of the dehydrin family are present in the nucleus, cytoplasm, and plasma membrane (Danyluk et al. 1998). We have found that two dehydrin-like proteins (dlps) accumulate in mitochondria of cereals in response to cold (Borovskii et al. 2000). We have also demonstrated a positive correlation between accumulation of the two mitochondrial dlps and cold-tolerance of the species studied. The aim of the study was to elucidate whether dehydrins penetrate mitochondria or they associate with the surface of the organelles.

Materials and Methods. Experiments were performed on etiolated winter wheat seedlings of the cultivar Irkutskaya ozimaya. Seedlings were grown on moistened filter paper at 22 C for 4 days. Mitochondria were isolated by differential centrifugation, as was described in (Welin et al. 1994). Mitochondria from wheat shoots were purified using a discontinuous Percoll gradient (De Virville et al. 1994). This gradient comprised 18, 23, and 35 % Percoll in the medium containing 0.3 mM sucrose, 40 mM Mops-KOH, pH 7.4, and 0.1 % BSA. Mitochondria were collected from the interface between 23 and 35 % Percoll. Purified mitochondria were used for protein isolation or they were treated with pronase E (1 mg/ml) at 37 C for 1 h in order to determine proteins localized at the outer membrane. Purified mitochondria were placed in the sample buffer, and proteins were extracted at 60 C for 15 min. Similar procedure was applied to the proteins from membrane fraction. SDS-PAGE was run in 10-12 % acrylamide gel in the modified Laemmli system (Laemmli 1970). Immunodetection was performed by the method of Timmons and Dunbar (1990). Polyclonal antibody against K fragment of dehydrins was kindly presented by T.J. Close (University of California, Riverside).

Results and Discussion. Earlier we found that dehydrins are able to associate with mitochondria very rapidly, as it was detected during seedling freezing (Borovskii et al. 2002). We supposed that these proteins did not penetrate inside the mitochondria but associated with their outer membrane. To verify this assumption, we used seedlings acclimated to 4°C for a week. Such a term for acclimation was chosen to permit dehydrins to be transported into mitochondria if such a transport could take place in the cell. In order to elucidate dehydrin localization, we treated isolated and purified mitochondria with pronase E. The results obtained permitted us to conclude that, during seedling cold acclimation, two dehydrins accumulated in mitochondria being localized at their outer membrane because they were accessible for protease (Figure 10). Earlier, outer localization of other protective proteins, maize low-molecular-weight heat-shock proteins, was detected in mitochondria in the test with protease (Borovskii and Voiniko 1993).

Dehydrins evidently fulfill a defensive function in the mitochondria. Understanding the mechanism of this defense is difficult, because it is unknown whether membrane lipids or proteins are the targets for dehydrin action. A dehydrin WCOR410 is known to associate with the plasma membrane. This dehydrin is supposed to protect this very frost-sensitive structure (Danyluk et al. 1998). Thomashow (1999) believes that dehydrin can suppress lipid phase transition from lamellar to hexogonal state, which implies its interaction with membrane lipids. Hara et al. (2003), relying on their data, suggested that dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress. Such type of protection is especially important for mitochondria because reactive oxygen species arise therein inevitably during respiration. Mitochondria convert about 2 % of consumed oxygen into hydrogen peroxide, which could damage membranes (Scandalios et al. 1997). On the other hand, dehydrin protective effects on proteins are not less significant. Dehydrin protection of some enzymes against denaturing was demonstrated in experiments in vitro (Wisniewski et al. 1999).

Thus, our results showed that the mitochondrial dehydrins are peripheral proteins and associated with the surface of the outer mitochondrial membrane. We suppose that dehydrins interact with membrane lipids and their stabilizing effect during cell dehydration is exerted via the retardation of lipid layer phase transition or due to reduction of lipid peroxidation. It seems likely that dehydrin association with mitochondria is an important mechanism of cold stress resistance.

References.

• Borovskii GB, Stupnikova IV, Antipina AA, Downs CA, and Voinikov VK. 2000. Accumulation of dehydrin-like-proteins in the mitochondria of cold-treated plants. J Plant Physiol 156:797-800.
• Borovskii GB, Stupnikova IV, Antipina AI, Vladimirova SV, and Voinikov VK. 2002. Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment. BMC Plant Biol http://www.biomedcentral.com/1471-2229/2/5.
• Borovskii GB and Voinikov VK. 1993. Localization of low-molecular heat-shock proteins at the surface and inside of maize mitochondria. Fiziol Rast (Moscow) 40:596-598 (Sov Plant Physiol, Eng Transl)
• Close TJ. 1997. Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol Plant 100:291-296.
• Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, and Sarhan F. 1998. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10:623-638.
• De Virville DJ, Aaron I, Alin MF, and Moreau F. 1994. Isolation and properties of mitochondria from Arabidopsis thaliana cell suspension cultures. Plant Physiol Biochem 32:159-166.
• Hara M, Terashima S, Fukaya T, and Kuboi T. 2003. Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217:290-298.
• Ismail AM, Hall AE, and Close TJ. 1999. Purification and partial characterization of a dehydrin involved in chilling tolerance during seedling emergence of cowpea. Plant Physiol 120:237-244.
• Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227:680-685.
• Scandalios JG. 1997. Oxidative stress and defense mechanisms in plants: introduction. Free Radic Biol Med 23:471-472.
• Thomashow MF. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571-599.
• Timmons TM, Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.
• Welin BV, Olson A, Nylander M, and Palva ET. 1994. Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 26:131-144.
• Wisniewski M, Webb R, Balsamo R, Close T.J, and Griffith M. 1999. Purification, immunolocalisation, cryoprotective and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiol Plant 105:600-608.

 

Association of dehydrins with membranes of winter wheat cells during cold treatmen. [p. 137-139]

I.V. Stupnikova, G.B. Borovskii, A.I. Antipina, and V.K. Voinikov.

Primary sites of cold injury in plants are cell membrane systems (Thomashow 1999). Therefore, protecting membrane structures is one of the important parts of efficient cold adaptation and tolerance mechanisms, in which cold-regulated (COR) proteins play significant role, functioning as cryoprotectors or regulators. Among COR-proteins, presumably protecting cell membranes the dehydrin family presents particular interest. Danyluk et al. (1998) found acidic dehydrins WCOR 410, a subtype of the dehydrin family, which are largely hydrophilic and have high content of charged residues and hydroxylated residues. WCOR 410 accumulates in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freezing-induced dehydration is likely to be more severe (Danyluk et al. 1998). Hara et al. (2003) showed that a dehydrin protein, purified from E. coli expressing citrus dehydrin cDNA prevented peroxidation of soybean liposomes in vitro. They suggested that the dehydrin facilitates plant cold acclimation by acting as a radical-scavenging protein to protect membrane systems under cold stress (Hara et al. 2003). We also found total cell dehydrin-like proteins with molecular weights of 209, 196, 169, 66, 50, and 41 kD, which are characteristic for hardening state of winter wheat plants (Stupnikova et al. 2000; Borovskii et al. 2002a), and mitochondrial proteins ranging from 52 to 63 kD depending on the species of cereals, which accumulated during cold adaptation (Borovskii et al. 2002b). Our next task was to elucidate whether and which dehydrins associate with other (nonmitochondrial) cell membranes at low temperature.

Materials and Methods. Experiments were performed on etiolated winter wheat seedlings of the cultivar Irkutskaya ozimaya. Seedlings were grown on moistened filter paper at 22 C for 4 days. For hardening, 3-day-old seedlings grown under optimal conditions were exposed to low temperature for 7 days at 4 C. Mitochondria were isolated by differential centrifugation, as was described in (Welin et al. 1994). Mitochondria from wheat shoots were purified using a discontinuous Percoll gradient (De Virville et al. 1994). Purified mitochondria were used for membrane protein isolation. Membranes were sedimented by ultracentrifugation at 105,000 g for 90 min. Membrane proteins were extracted with sample buffer at 50-60 C for 20 min and separated in 12 % SDS-PAAG. Proteins were transferred to the nitrocellulose membrane and detected with antibody against dehydrins. Immunodetection was performed by the method of Timmons and Dunbar (1990). A polyclonal antibody against the K fragment of dehydrin was kindly provided by T.J. Close (University of California, Riverside).

Results and Discussion. To study dehydrins associated with other (nonmitochondrial) cell membranes at low temperature, we isolated the fractions of such cell membranes as tonoplast and plasma membrane from wheat seedlings exposed to low temperature for 7 days and from seedlings grown at 22 C (control). Among wheat membrane proteins, we detected four dehydrins with molecular weights of 209, 196, 66, and 50 kD (Figure 11). Dehydrin-like proteins with similar molecular weights were found earlier among total cell water-soluble dehydrins, isolated from winter wheat seedlings and crowns (Borovskii et al. 2002a; b). The main part of these proteins has membrane localization. Both total cell and membrane dehydrins accumulated during cold adaptation (Figure 11), what taking together with their hydrophilic and heat-stable nature allows suggesting that they protect and stabilize membranes structures of wheat cells during cold stress. We also believe that a limited number of dehydrin types could exert stabilization of diverse membrane structures, which could be injured by cold. Other dehydrin-like proteins (Borovskii et al. 2002a) revealed in total cell fraction possibly are water-soluble proteins protecting possibly cold-sensitive cell proteins. Two membrane dehydrin groups (66 and 50 kD) had molecular weights close to those of mitochondrial dehydrins (63 and 52 kD). The methods used did not permit us to decide whether these proteins with close molecular weights were similar or different proteins. Therefore, so far we can not answer the question whether dehydrins associated with mitochondria can be detected in other cell compartments. On the basis of the data we may conclude that dehydrins with molecular weights 209, 196, 66, and 50 kD could interact with lipids of cell membrane structures during cold treatment and stabilize them preventing lipid phase transition.

References.

• Borovskii GB, Stupnikova IV, Antipina AI, and Voinikov VK. 2002a. Accumulation of dehydrins and RAB-proteins in wheat seedlings during low temperature adaptation. Rus J Plant Phys 49:1-7.
• Borovskii GB, Stupnikova IV, Antipina AI, Vladimirova SV, and Voinikov VK. 2002b. Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment. BMC Plant Biol http://www.biomedcentral.com/1471-2229/2/5.
• Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, and Sarhan F. 1998. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10:623-638.
• De Virville DJ, Aaron I, Alin MF, and Moreau F. 1994. Isolation and properties of mitochondria from Arabidopsis thaliana cell suspension cultures. Plant Physiol Biochem 32:159-166.
• Hara M, Terashima S, Fukaya T, and Kuboi T. 2003. Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217:290-298.
• Stupnikova IV, Borovskii GB, Dorofeev NV, Peshkova AA, and Voinikov VK. 2002. Accumulation and disappearance of dehydrins and sugars depending on freezing tolerance of winter wheat plants at different developmental phases. J Therm Biol 27:55-60.
• Thomashow MF. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann Rev Plant Physiol Plant Mol Biol 50:571-599.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.
• Welin BV, Olson A, Nylander M, and Palva ET. 1994. Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 26:131-144.

 

Identification of HMW and LMW subunits of glutenin and their impact on various technological parameters. [p. 139-143]

E.V. Berezovskaya, V.A. Trufanov, T.N. Mitrofanova, and L.S. Kazmiruk.

Wheat reserve proteins gliadin and glutenin are the key components of gluten, which are responsible for rheological and baking qualities of wheat flour. Glutenins belong to the class of polymeric proteins, whose individual polypeptides are known as high- and low-molecular weight subunits (HMW- and LMW- glutenin subunits). Subunits of these two groups differ in amino-acid composition, molecular mass (from 23 to 68 kDa for LMW-glutenin subunits and from 77 to 160 kDa for HMW-glutenin subunits), and structure (Kasarda 1999). HMW-glutenin subunit genes are located on the long arms of chromosomes 1A, 1B, and 1D, loci Glu-A1, Glu-B1, and Glu-D1, respectively (Payne 1987). LMW-glutenin subunit genes are on the short arms of chromosomes 1AS, 1BS, and 1DS (Singh et al. 1988). Many researchers have shown that allele variations of HMW- and LMW-glutenin subunits are associated with differences in technological parameters of flour and dough (Payne 1987; Autran et al. 1987; Gupta et al. 1989). Both LMW- and HMW-glutenin subunits, whose number is normally three-four times that of LMW-glutenin subunits, make a significant impact on viscose-elastic properties of dough and other technological parameters of flour (Pogna et al. 1990; Metakovsky et al. 1997). Variations in the composition of these subunits among different genotypes are in fact the major factor, which determines differences in baking qualities of dough and bread.

This work summarizes the results of complex investigation of grain reserve protein, glutenin, in connection with productivity and quality of gluten of two unrelated cultivars of soft spring wheat and four hybrid F9 and F10 from their interbreeding, establishment of the impact of various alleles coding glutenins, and the results of quantitative determination of HMW- and LMW-glutenin subunits and their correlation and role in determination of rheological properties.

Materials and Methods. The objects of investigation were the varieties of soft spring wheat Rollo (R) and Drott (D) and four constant hybrid forms from their interbreeding: RxD-I, RxD-II, RxD-III, and RxD-IV in F9 and F10 generations. The spring wheat cultivar Drott (Fylgia II/Sviöf 0990) has high resistance to fungal diseases, it is a highly productive variety with fairly strong straw. The Early Tyumenskaya cultivar was selected by mass selection method from the cultivar Rollo (K-45657, Norway).

For glutenin extraction, we used a Tris-HCl buffer, pH 6.8 containing sodium dodecylsulphate and 10 % b-mercaptoethanol following complete gliadin extraction by 70 % ethanol. Restoration of S-S-bonds was conducted for 15 hours at 40 C. SDS-PAAG electrophoresis was performed according to Laemmli (1970), overnight, at constant current strength and stable voltage. Concentrating and separating gels (3 and 10 %, respectively), containing acrylamide, methylenbisachrilamide, Tris-HCl (pH 6,8), TEMED, ammonium persulphate, and 10 % SDS. The gels were scanned and density-measured. A standard protein mixture was used to determine molecular mass (Mr) of glutenin subunits. HMW-glutenin subunits were identified according to Payne et al. (1983). Aggregation ability of gluten proteins, as one of the major characteristics of gluten rheological properties, was determined by the modified method of Arakawa and Yonezawa (1975), which is widely used for classifying cultivars by gluten quality. The tests were conducted directly with flour and gluten washed in 1 % NaCl solution and in distilled water. Washed gluten was extracted by 0.01 M acetic acid for 16-20 h at 4 C with constant agitation. The extracts then were centrifuged at 7,000 g for 15 min. Protein concentration was determined by spectrophotometric Calcar's method and calculated as per the following formula:

1,45 D280 - 0,74 D260 = C mg/ml,
where 0.74 and 1.45 = recalculation ratios,
C = protein concentration in the solution,
D280 and D260 = optical solution density.

Aggregating ability was determined by the change of absorption at turbidity increase, for 10 min with the interval of 30 sec, protein solution + 0.2 M phosphate buffer, pH 5.65, (Arakawa and Yonezawa 1975) or pH 9.5

Trufanov 1994) containing 2 M NaCl. The constant of aggregation speed K was calculated at the first stages of aggregation by the turbidity change using the following equations:

K = r/C4, r = t3/3t, where
K = constant of the initial aggregation stage;
t = solution turbidity index;
t = time in sec;
C = protein concentration, %; and
t10/C = aggregation constant after 10 min of processing, calculated per unit of protein concentration in the solution.

Result and Discussion. Electrophoresis of proteins from standard cultivars (Hope, Bezostaya (unawned) spring, Novosibirskaya 67, and Chinese Spring) allowed the identification of high-molecular-weight subunits according to Payne (1983). The genes encoding glutenin HMW-glutenin subunits are distributed on the long chromosome arms 1A, 1B, and 1D. Three loci, Glu-A1, Glu-B1, and Glu-D1, jointly make up a locus i and encode HMW subunits. Two subunits of loci Glu-B1 and Glu-D1 have two subgroups x and y, subunits differing in mobility and amino-acid composition, including cystein content. Standard cultivars with identified subunits (Table 6) allowed us to establish the position of each subunit via comparison of densitograms (Figure 12) and Rf in the course of electrophoresis in SDS-PAAG. We also used alternative identification system using molecular masses (Ng and Bushuk 1989) (Table 7).

Table 6. Genotype of high-molecular-weight glutenin subunits of some standard wheat cultivars.

Chromosome arm	Locus	HMW-glutenin subunit composition
		Novosibirskaya 67	Bezostaya	Hope	Chinese Spring
1AL	Glu-A1	a (1)	b (2*)	a (1)	c (null)
1BL	Glu-B1	b (7+8)	c (7+9)	c (6+8)	b (7+8)
1DL	Glu-D1	a (2+12)	---	d (5+10)	a (2+12)

Table 7. Molecular weight of the glutenin subunits of some standard wheat cultivars.

Subunit	Molecular weight
1	114.7
2*	106.6
2	103.7
5	98.5
6	86.6
7	84.9
8	75.8
9	72.8
10	71.8
12	69.3

The data on the composition and molecular weight of functional glutenin subunits and density-measuring scanning of electrophoretic spectra of the standard wheat cultivars show that there were identified hypothetic genotypic formulas for HMW-glutenin subunits of the cultivars Rollo and Drott and their four hybrids. We demonstrated redistribution of inherited information between hybrids (Table 8).

Table 8. Allelic variation in glutenin subunits in the cultivars Rollo and Drott and their hybrids.

Chromosome arm	Locus	Rollo	Drott	Rollo/Drott hybrid
				I	II	III	IV
1AL	Glu-A1	c (null)	a (1)	a (1)	c (null)	a (1)	a (1)
1BL	Glu-B1	b (7+8)	c (6+8)	c (6+8)	c (6+8)	b (7+8)	b (7+8)
1DL	Glu-D1	a (2+12)	d (5+10)	d (5+10)	a (2+12)	d (5+10)	a (2+12)

All the four hybrids produced by crossing of two parental cultivars Rollo and Drott have significant differences in their HMW-glutenin subunits. Thus, the I hybrid completely correspond to the Drott HMW-subunits, but manifests differences in the realm of LMW-glutenin subunits (Figure 12). In the other three hybrids, parental HMW-glutenin subunits were redistributed: the 'Rolo/Drott' hybrid II inherited from Rollo subunits 2+12 of locus Glu-D1 and nonexpressing subunit of null-locus Glu-A1, and from Drott subunits 6+8 of locus Glu-B1; 'Rolo/Drott' hybrid III is closer by component composition to Drott , but is different from it by the single subunit 7, typical of Rollo and belonging to locus Glu-B1; 'Rolo/Drott' hybrid IV is closer to Rollo, differing by one subunit (1) encoded by locus Glu-A1 (Table 8).

Subunit 1 is associated with high baking quality in contrast to the null allele. This subunit might possess a unique primary structure, which participates in the formation of stable gluten aggregates. Subunits 5+10 also correlate with good baking quality, in contrast to subunits 2+12. Differences in amino-acid sequence and secondary structure between allele subunits 5+10 and 2+12 also were revealed (Anderson 1996). We identified genotypic formulas that contain both 'good', 5+10 and 1 (in Drott), and 'bad' subunits, 2+12 (in Rollo).

Apparently, Drott is likely to produce higher dough quality than Rollo, which is confirmed by aggregation and technological qualities parameters. 'Rolo/Drott' hybrids I and III might also yield good parameters, as their spectrum contains HMW-glutenin subunits 5+10 obtained from Drott. We assumed that 'Rolo/Drott' hybrid IVwill manifest poor baking qualities, because it inherited subunits 2+12 from both parents, which do not have good parameters of technological properties.

High-molecular-weight subunits represent only 10 % of all the gluten proteins, but their role in determining baking quality in accordance with different subunit types is immense. Another important factor in determination and more precise definition of various technological parameters is the second group of polymeric proteins LMW-glutenin subunits. HMW- and LMW-glutenin subunits differ in the values of amino-acid composition, molecular weight (from 23 to 68 kDa) and structure (Kasarda 1999).

The quantity of LMW-glutenin subunits exceeds that of the HMW-glutenin subunits by 3-4 times (Kasarda 1999). Their characterization is complicated, because they are controlled by a large number of genes and their subunits are often insoluble after restoration of intermolecular disulfide bonds.

To investigate their participation in the characterization of the cultivars, we determined their relative quantity and calculated the proportion between the quantities of LMW- and HMW-glutenin subunits. According to Hou et al. (1996), this proportion in flour proteins may act as an important parameter in the evaluation of rheological and baking qualities of wheat.

Quantitative determination of the LMW- and HMW glutenin subunits was conducted with the help of density-measuring analysis DDS-PAAG of electrophoretogram (Figure 13). Quantitative content of HMW- and LMW-glutenin subunits was calculated with the help of software; the results are presented in relative units (Table 9). The data demonstrate the fact that LMW-glutenin/HMW-glutenin subunit proportion is significantly higher in Rollo (2.95) compared to Drott and lower in their hybrids (3.0-3.23, Table 9). This value has a positive correlation with the data on the flour sedimentation analysis: The higher the LMW-glutenin/HMW-glutenin subunit index, the higher the sedimentation parameters. Rollo has a much higher index (2.95) than that of Drott, with a high sedimentation index (42 ml). So, we see a tight correlation of LMW-glutenin/HMW-glutenin subunit proportion and parameters of sedimentation flour analysis. Hybrids do not manifest significant differences in these parameters (Table 10).

Table 9. Percentage of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits in the cultivars Rollo and Drott and their hybrids.

Line	HMW-GS	LMW-GS	LMW-GS/HMW-GS
Rollo	25.5	75.4	2.95
Drott	46.4	53.6	1.16
Rollo/Drott I	30.5	69.5	2.27
Rollo/Drott II	23.6	76.4	3.23
Rollo/Drott III	25.0	75.0	3.00
Rollo/Drott IV	24.7	75.3	3.04

Table 10. Technological properties and aggregation parameters of Rollo and Drott and four 'Rollo/Drott' hybrids. Data is from 2001; t10/C - aggregation constant at the 10th minute of the process.

	Rollo	Drott	Hybrid I	Hybrid II	Hybrid III	Hybrid IV
Technological parameter	c (null)	a (1)	a (1)	c (null)	a (1)	a (1)
	b (7+8)	c (6+8)	c (6+8)	c (6+8)	b (7+8)	b (7+8)
	a (2+12)	d (5+10)	d (5+10)	a (2+12)	d (5+10)	a (2+12)
LMW-GS/HMW-GS	2.95	1.16	2.27	3.23	3.00	3.04
aggregation constant	4.84	12.63	13.60	8.91	13.51	5.90
t10/C	30.26	26.11	28.68	34.82	39.57	33.09
sedimentation (ml)	42	29	35	36	29	31

Comparative investigation of aggregating ability of the vinegar-soluble fraction of flour reserve proteins of hybrids in the F9 and F10 with various types of electrophoretic spectra showed that among the biotypes under study, the ratio of initial aggregation stage (K) ranges between 4.84-13.6. Genotypes, whose electrophoretic spectrum contains glutenin subunits 6+8 (Drott and hybrids I and II), regardless of the presence of other subunits, had aggregation ratio 12.63, 8.91, and 13.6, respectively; with the final aggregation stage index (t10/C) equaling 26.11, 28.68, and 34.82, respectively.

Thus, we performed a complex investigation of the grain-reserve protein glutenin. We identified correlations between the composition of HMW-glutenin subunits, the proportion of HMW- and LMW-glutenin subunits, and a number of technological properties. HMW-glutenin subunit 1 was shown to be associated with high baking quality, in contrast to the null allele. Subunits 5+10 also correlate with high baking qualities in contrast to subunits 2+12.

References.

• Anderson O, Bekes F, and Kuhl J. 1996. Use of bacterial expression system to study wheat high-molecular-weight (HMW) glutenins and the construction of synthetic HMW-glutenin genes. Cereal Chem 195-198.
• Arakawa T and Yonezawa D. 1975. Agr Biol Chem 39(11):2123-2128.
• Autran JC, Laignetet B, and Morel MY. 1987. Characterisation and quantification of low molecular weight glutenins in durum wheat. Biochimie 69:699-711.
• Gupta RB, Singh NK, and Shepherd KW. 1989. The cumulative effect of allelic variation in LMW andYMW glutenin subunits on dough properties in the progeny of two bread wheats. Theor Appl Genet 77:57-64.
• Hou G , Yamamato H, and Ng PKW. 1996. Relationships of quantity of glutenin subunits of selected us soft wheat flours to rheological and baking properties. Cereal Chemistry 73(3):358-363.
• Kasarda DD 1999. Glutenin polymers: The in vitro to in vivo transition. Cereal Food World. 44:566-571.
• Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the bacteriophage Nature 4:277:680-685.
• Metakovsky EV, Felix J, and Branland G. 1997. Association between dough quality and certain gliadin alleles in French common wheat cultivars. J Cereal Sci 26:371-373.
• Ng PKW and Bushuk W. 1988. Statistical relationships between high molecular weight of glutenin and breadmaking quality of Canadian-grown wheats. Cereal Chem 65:408-413.
• Payne PI. 1987. Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Ann Rev Plant Physiol 38:141-153.
• Payne PI and Lawrence GI. 1983. Catalogue of alleles for the complex loci, Glu-A1, Glu-B1, and Glu-D1 which coded for high-molecular-weight subunits of glutenins in hexaploid wheat. Cereal Res Commun 11:29-35.
• Pogna PE, Autran JC, Mellini F, Lafiandra P, and Feillet P. 1990. Chromosome 1B-encoded gliadins and glutenin subunits in durum wheat: genetics and relationship to gluten strength. J Cereal Sci 11:15-34.
• Singh NK and Shepherd KW. 1988. Linkase mapping of the genes controlling endosperm proteins in wheat. 1. Genes on the short arms of group-1 chromosomes. Theor Appl Genet 75:628-641.
• Trufanov VA. 1994. Wheat Gluten - Quality Problems. Novosibirsk: Nauka. P. 167.

 

Subunits of functional glutenin as structural elements of complex gluten proteins. [p. 143-147]

E.V. Berezovskaya, V.A. Trufanov, T.N. Mitrofanova, and T.A. Pshenichnikova.

Native wheat glutenin is a complex set of biochemically diverse polypeptides connected by different forces of protein-protein interactions. The interest in identifying peculiarities in the genetically determined composition of functional glutenin subfractions is because of their immediate participation in the formation of a gluten-protein complex that is responsible for baking properties in flour. High-molecular-weight glutenin subunits of the majority of industrial wheat cultivars of many countries have been well-studied. The participation of these structural elements in the formation of compositionally complicated, multicomponent protein-gluten complex responsible for dough and bread quality are known.

Glutenin is known to consist of at least 15-17 subunits with different molecular mass, amino-acid composition, and primary and secondary structure are associated into a single permolecular protein complex via intermolecular disulfide bonds stabilizing a three-dimensional structural gluten matrix. The number of these bonds is a genotypically determined trait of a genetically specific character that determines gluten physical properties and dough rheological characteristics (Trufanov 1994).

To find favorable alleles, we were interested in the impact of chromosomes of homoeologous groups 1 and 6, which control storage protein synthesis, in wheat lines with intervarietal substitutions. These lines have contrasting baking qualities based on the quantitative content of HMW-glutenin subunits present.

Materials and Methods. Soft wheat lines with intervarietal substitutions of chromosomes 1A, 1B, 1D, 6A, 6B, and 6D of the cultivar Novosibirskaya 67 (N67), a strong wheat, and the high-protein recipient cultivar Diamant 1 (Dm) with low technological quality were used (Obukhova et al. 1997; Maystrenko et al. 1993).

Glutenin subfractions (GN) were obtained from freshly ground flour after removal of albumins, globulins, and gliadins by successive extraction with 0.05 M acetic acid (subfraction GN-1), 4 M urea (subfraction GN-2), and 4 M urea in the presence of 2-mercaptoethanol (subfraction GN-3) (Trufanov 1994). Protein fractions were dialyzed against 0.01 M acetic acid and lyophilized. Subunits composition in the subfractions GN-1, GN-2, and GN-3 was studied after SDS electrophoresis in 9 % polyacrylamide gels according to Laemmli (1970). Evaluation of quantitative content of PAGE zones in glutenin subfractions of each substituted line fraction was by densitometric analysis of electrophoretograms (Figure 13). Quantitative content of five HMW-glutenin subunits was calculated with the help of a computer program. The results were expressed in units and in % relative to the recipient cultivar Dm (Figure 14).

Result and Discussion. The donor cultivar N67 exceeds the recipient Dm in relative content of subunits 2 and 3 in the easily soluble GN-1 fraction (by 10-12 %); in sparingly soluble GN-2 fraction, subunits 4 and 5 by 13 and 45 %, respectively; and in the insoluble (without restoration of SS bonds) GN-3 fraction, subunits 1 and 2 by 25 and 10 %, respectively) (Figure 14). Simultaneously, the donor N67 is inferior to the recipient Dm in terms of GN-1 content of subunits 1 and 4 by 11 and 20 %, respectively; in GN-2, subunits 1 and 3 by 17 and 7 %, respectively; and in GN-3, subunits 3, 4, and 5 by 5, 20, and 36 %, respectively.

Obukhova et al. (1997) and Maystrenko et al. (1993) showed that in Dm and N67, subunits 1 and 2 are controlled by genes on chromosomes 1A, 1B, and 1D, whereas subunit 3, which contains three components, is controlled by genes on chromosomes 1B and 1D. One component of subunits 3 belongs to N67 and is controlled by genes on chromosome 1B; the second belongs to Dm and also is controlled by genes on 1B; and the third component, common for both cultivars, is controlled by genes on chromosome 1D. Several authors (Obukhova et al. 1997; Maystrenko et al. 1993; Payne 1979, 1997) assume that chromosomes of different homoeologous groups participate in genetic control of compositionally complicated subunits 4 and 5 (Figure 15).

Compared to Dm, subunit 1 prevails in the subfraction GN-1 in N67-Dm DS1A DS6A, and DS6B; in subfraction GN-2 in Dm-N67 DS1B; and in subfraction GN-3 in Dm-N67 DS1B, DS1D, DS6A, and DS6B. Subunit 2 prevails in Dm-N67 DS6A in the GN-1 subfraction, in all substituted lines of the GN-2 subfraction, and in Dm-N67 DS1A, DS1B, and DS6A in subfraction GN-3. Subunit 3 content was higher only in Dm-N67 DS6A in subfraction GN-1 and in Dm-N67 DS6B and Dm-N67 DS6D. Subunit 4 content was higher in all the lines of subfractions GN-1 and GN-3. Subunit 5 prevailed only in the subfraction GN-1 in substituted lines with chromosomes 1D and 6A and was slightly higher in Dm-N67 DS1A in subfraction GN-3.

The positive influence of the subunit content on glutenin in the recipient cultivar Dm was to varying degrees caused by chromosomes from the donor cultivar N67. A considerable substitution effect was evident in the case of chromosome 6A. Substitution of this chromosome resulted in intensifying synthesis of all the subunits in soluble subfraction GN-1, two in the sparingly soluble subfraction GN-2, and two in the insoluble fraction GN-3. Extraction of GN-3 subunits is possible only after complete restoration of inter- and intramolecular SS links that stabilize the gluten structural matrix. Simultaneously, the content of individual subunits in these substitution lines was lower than in the recipient, particularly that of subunits 3, 4, and 5.

Differences in solubility of subfractions GN-1, GN-2, and GN-3 are known to be associated with the density of their spacial structure, which determines the ability of these proteins to form permolecular protein associates, characteristic of gluten (Trufanov 1994). Formation of protein glutenin macroassociates in the grains in vivo happens with the participation of various intermolecular forces: ion-electrostatic interactions conditioned by acidic and basic amino acids (AA), hydrophobic contacts (hydrophobic AA), and SS links (cystine). Consequently, the combined impact of nonvalent and covalent forces are determined by the quantity and biochemical properties of individual polypeptides (subunits), primarily by the content and location in polypeptide chains of reactive SH groups that are able to form intermolecular SS bonds. The folding of proteins of various origin and the chaperon-dependent assembling of protein macroassociates in the cell in vivo are known to happen in cotranslation and/or posttranslation periods and to be catalyzed by the system of SH/SS-metabolism enzymes, in particular, by protein disulfide isomerase (Chi-Chen Yong 1998; Marusich 1998; Fisher 1998) responsible for formation, splitting, and isomerization of SS bonds in the proteins. The significant changes observed in the substitution lines in the quantitative content and proportion of HMW-glutenin subunits in functional glutenin fractions of contrasting in technological properties wheat varieties may play an important role in the formation (assembling) and stabilization of functionally important glutenin complexes as a structural basis of gluten.

References.

• Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the bacteriophage. Nature 4:277:680-685.
• Marusich EI, Kurochkina LP, and Mesyanzhinov VV. 1998. Chaperons in the bacteriophage assembling T4. Biochem 63(4):473-482.
• Maystrenko OI, Ermakova MF, and Popova RK. 1993. Effects of some chromosome substitutions on wheat Triticum aestivum L. technological properties. In: Proc II Russian Symp New Methods in Pant Biotechnology, 18-20 May. Pushino, p. 199.
• ObukhovaLV, Maystrenko OI, Generalova GV, Ermakova MF, and Popova RK. 1997. Composition of high-molecular weight glutenin subunits in common wheat substitution lines created from cultivars with contrasting bread making qualities. Genetica 33:1179-1184.
• Payne PI, Corfield KG, and Blackman JA. 1979. Identification of a high-molecular-weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theor Appl Genet 5:153-159.
• Payne PI, Nightingale A, Krattiger AF, and Holt LM. 1997. The relationship between HMW glutenin subunit composition and the bread-making quality of British-grown wheat varieties. J Sci Food Agric 40:51-65.
• Trufanov VA. 1994. Wheat Gluten - Quality Problems. Novosibirsk: Nauka 167 p.
• Yong CC. 1998. Isomerase and chaperon activities of protein disulfide isomerase are indispensable for its functioning as foldase. Biochem 63(4):484-490.
• Fisher MT. 1998. Participation of GroE chaperonin in folding and assembling of dodekamer glutaminsythetase. Biochem 63(4):453-472.

 

Specific lipoxygenase activity in Triticum aestivum subsp. aestivum / Aegilops speltoides introgression lines. [p. 147-148]

M.D. Permyakova, V.A. Trufanov, A.V. Permyakov, and T.A. Pshenichnikova (Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russian Federation).

Lipoxygenase (linoleat:oxygen oxidoreductase, EC 1.13.12, Lpx) is known to catalyze the oxidation of unsaturated fatty acids resulting in formation of peroxide and hydroperoxide compounds. This enzyme is widely distributed in plant cells. Lipoxygenase reactions may initiate the synthesis of signaling molecule or be involved in inducing structural or metabolic changes in the cell (Siedow 1991; Grechkin 1999).

Our research involved T. aestivum subsp. aestivum / Ae. speltoides introgression lines. Species belonging to the genus Aegilops, a wild relative of wheat, are widely used for introducing agriculturally important traits, such as resistance to fungal diseases and pests and tolerance to high salinity and temperature, into the genome of common wheat. The common spring wheat Rodina was used as the female parent in producing the introgression lines. This line is part of the 'Arsenal' collection produced by I.F. Lapochkina (Institute of Agriculture of Central Regions in Non-Chernozem Zone of Russia, Nemchinovka and Lapochkina 2001).

Lipoxygenase activity was analyzed in Tris-soluble flour extracts according to Doderer et al. (1992). The average values of specific Lpx activity in the parental cultivars and eight introgression lines for 2 years are in Figure 16. Enzyme activity is a level lower in wild cereals than that in wheat lines. This difference possibly is because Lpx genes are in triplicate in hexaploid wheat, although this point requires further study. Introgression causes a significant increase in Lpx values in lines 32W, 75W, 84W, 178i, and 170i in a genetic background of Rodina. Lipoxygenase activity in lines 167i, 182i, 255i, and 176i significantly decreased in comparison with Rodina.

Previously, the same introgression lines was characterized by means subtelomeric repeats Spelt 1 and Spelt 52 being specific markers Ae. speltoides (Salina et al. 2001). We have found that values of specific Lpx activity are directly connected to Spelt 1 amount (Figure 17). Any connection Lpx with Spelt 52 was not revealed.

Alien introgression also can lead to change of Lpx activity, which is connected with physiological processes in plants. Lines with alien chromosomes may be a source of certain disease-resistance genes and other traits that change a plants adaptive potential. These transfers into common wheat cultivars may be useful for breeding programs.

References.

• Doderer A, Kokkelink I, Van der Veen S, Valk BE, Schram AW, and Douma AC. 1992. Purification and characterization of two lipoxygenase isoenzymes from germinating barley. Biochim Biophys Acta 1120:97-104.
• Grechkin A. 1998. Recent development in biochemistry of the plant lipoxygenase pathways. Prog Lipid Res 37:317-352.
• Lapochkina IF. 2001. Genetic diversity of "Arsenal" collection and its use in wheat breeding. Abstr Internat Appl Sci Conf 'Genetic Resources of Cultural Plants'. 13-16 November, 2001, St. Petersburg, Russian Federation. Pp. 133-135.
• Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275.
• McDonald CE. 1979. Lipoxygenase and lutein bleaching activity of durum wheat semolina. Cereal Chem 56:84-89.
• Salina EA, Adonina IG, Efremova TT, Lapochkina IF, and Pshenichnicova TA. 2001. The genome-specific subtelomeric repeats for study of introgressive lines T. aestivum x Ae. speltoides. EWAC Newslet, Novosibirsk, Russian Federation. Pp. 161-164.
• Siedow JN. 1991. Plant lipoxygenase: structure and function. Ann Rev Plant Physiol Plant Mol Biol 42:145-188.

 

Specific lipoxygenase activity in intervarietal substitution lines of hexaploid wheat. [p. 148-150]

M.D. Permyakova, V.A. Trufanov, A.V. Permyakov, and T.A. Pshenichnikova (Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russian Federation).

Lipoxygenases (Lpx, EC 1.13.11.12) are nonheme, iron-containing dioxygenases widely distributed in plants, fungi, and animals. Lipoxygenase catalyzes the addition of molecular oxygen to polyunsaturated fatty acids to produce an unsaturated fatty acid hydroperoxide. Products of the Lpx pathway are involved in inducing structural or metabolic changes in the cell. In plants, Lpx has been associated with some processes in number of developmental stages (Siedow 1991) and with mobilization of storage lipids during germination (Feussner et al. 2001). Lipoxygenase also is used as a storage protein during vegetative growth (Tranbarger et al. 1991). Lipoxygenase also influences quality parameters of wheat gluten (Mc Donald 1979; Shiba et al. 1991).

Lipoxygenase expression is regulated by different effectors such as the source/sink status (Feussner et al. 2001), jasmine acid (Park et al. 1994) and abscisic acid (Melan et al. 1993), and also by different forms of stress, such as wounding, water deficiency, or pathogen attack (Porta et al. 1999; Melan et al. 1993).

Lines with substitutions of individual chromosome pairs of the homoeologous group in wheat are convenient genetic material for the study of donor and recipient gene effects on their expression and enzyme specific activity. The objective of our research is the substitution lines of Saratovskaya 29-Janetskis Probat (S29-JP) for all chromosomes excluding 1. These lines were developed at the Institute of Cytology and Genetics, Novosibirsk, Russian Federation.

In order to measure Lpx activity, proteins were extracted from the flour by Tris buffer 1:1 (w:v) at 4 C. Lipoxygenase activity was assayed spectrophotometrically under the wavelength 234 nm (Doderer et al. 1992). One unit of activity was defined as the change in optical density on 0.001/min. Specific activity was expressed by ratio of activity units to 1 mg of protein in 1 ml of incubation media. Protein concentration was determined by according to Lowry et al. (1951).

Results of Lpx activity measurement in all investigated lines are shown in Figure 18. The diagrams present average data of the three biological and three analytical replicates. The donor JP exceeded the recipient S29 by 60 % for the specific activity of Lpx. In all lines excluding 3D and homeologous group 5, this trait is significantly higher than that of the donor parent JP.

The genes responsible Lpx synthesis are located on the chromosomes of the homeologous groups 4 and 5 (Hart and Langstone 1977). Judging from our results, chromosomes of different homoeologous groups participate in control of this character. We have shown that intervarietal substitution of the homeologous groups 1 and 6 chromosomes affect the functional activity of this enzyme (Trufanov et al. 2001). From this data, we concluded that there are genes or regulators of Lpx activity along with structural genes.

The data from 3 years of Lpx activity measurements are in Figure 19. Levels of Lpx change over different years, but Lpx activity inheritance in the parental cultivars and the substitution lines is similar during the experimental period. Average data for the 3-year experiment with this set are given in Figure 20. The most significant influence on enzyme specific activity is from the substitution of chromosome 4A. Substitution of chromosomes from homeologous group 5 does not change Lpx activity. Probably, chromosomes from JP have such Lpx alleles that cannot compensate for the activity of corresponding alleles of S29. The chromosomal location of Lpx structural genes is known, but the regulation of its activity may be under separate genetic control. Taking into account the important physiological role of Lpx, further genetic studies will be extremely important.

References.

• Doderer A, Kokkelink I, Van der Veen S, Valk BE, Schram AW, and Douma AC. 1992. Purification and characterization of two lipoxygenase isoenzymes from germinating barley. Biochim Biophys Acta 1120: 97-104.
• Feussner I, Kühn H, and Wasternack C. 2001. Lipoxygenase-dependent degradation of storage lipids. TRENDS Plant Sci 6:268-273.
• Hart GE and Langstone PJ. 1977. Chromosome location and evolution of isozyme structural genes in hexaploid wheat. Heredity 39:263-277.
• Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275.
• McDonald CE. 1979. Lipoxygenase and lutein bleaching activity of durum wheat semolina. Cereal Chem 56:84-89.
• Melan MA, Dong X, Endara ME, Davis KR, Ausubel FM, and Peterman TK. 1993. An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acids, and methyl jasmonate. Plant Physiol 101:441-450.
• Park TK, Holland MA, Laskey JG, and Polacco JC. 1994. Germination- associated lipoxygenase transcripts persist in maturing soybean plants and are induced by jasmonate. Plant Sci 96:109-117.
• Porta H, Rueda-Benitez P, Campos F, Colmenero-Flores JM, Colorado JM, Carmona MJ, Covarrubias AA, and Rocha-Sosa M. 1999. Analysis of lipoxygenase mRNA accumulation in the common bean (Phaseolus vulgaris L.) during development and under stress condition. Plant Cell Physiol 40:850-858.
• Shiiba K, Negishi Y, Okada K, and Nagao S. 1991. Purification and characterization of lipoxygenase isozymes from wheat germ. Cereal Chem 68:115-122.
• Siedow JN. 1991. Plant lipoxygenase: structure and function. Ann Rev Plant Physiol Plant Mol Biol 42:145-188.
• Tranbarger TJ, Franceschi VR, Hildebrand DF, and Grimesa HD. 1991. The soybean 94-kilodalton vegetative storage protein 1s a lipoxygenase that 1s localized in paraveinal mesophyll cell vacuoles. Plant Cell 3:973-987.
• Trufanov VA, Osipova SV, Permyakova MD, Mitrofanova TN, Pshenichnikova TA, and Maystrenko OI. 2001. SH/SS metabolism enzyme activity and gluten quality of wheat lines with intervarietal substitution of the 1 and 6 homeologous groups of chromosomes. EWAC Newslet, Novosibirsk. Pp. 176-180.

 

 

 

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.

 

Glycine-rich antimicrobial peptides from seeds of Triticum kiharae Dorof. et Migusch. [p. 150-151]

T.I. Odintsova and V.A. Pukhalskiy (Vavilov Institute of General Genetics) and A.K. Musolyamov and Ts.A. Egorov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry).

Peptides with antimicrobial activity are present in most plant species. Most plant antimicrobial peptides isolated so far contain even numbers of cysteine residues (4, 6, or 8) forming disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level and cysteine motifs, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and hevein- and knottin-type antimicrobial peptides (Garcia-Olmedo et al. 1998). All antimicrobial peptides studied so far exert their antimicrobial activity at the level of the plasma membrane of the target microorganism, but different peptide types are likely to operate via different mechanisms. In a number of plant species, a considerable induction of genes expressing antimicrobial peptides has been observed upon infection with pathogens suggesting their role in inducible defense response of plants. Constitutive expression of heterologous antimicrobial peptide genes, which has resulted in enhanced resistance to particular microbial pathogens, has been achieved for several plant species. New potent antimicrobial peptides are of considerable interest for production of transgenic resistant crops, they also have a considerable potential as novel therapeutic agents, disinfectants and food preservatives. Considerable progress has been made recently in the identification of new antimicrobial peptides in different plant species. However, wild relatives of wheat and related species highly resistant to pathogenic microorganisms are poorly studied. In this work, we analyzed the peptide composition of seeds of Triticum kiharae, a synthetic allopolyploid produced by crossing T. timopheevii subsp. timopheevii with Ae. tauschii, which is highly resistant to most fungal pathogens.

Materials and Methods. The peptide fraction was extracted from T. kiharae flour with acids (1 % trifluoroacetic acid, 1 M HCl, and 5 % HCOOH) for 1 h at room temperature and precipitated overnight with cold acetone, redissolved, and subjected to chromatography on Heparin Sepharose. Proteins and peptides were eluted with a stepwise NaCl gradient. The unbound, 100- and 500-mM NaCl fractions were desalted on a C8 cartridge, dried on a Speedvac concentrator, and separated by size-exclusion chromatography on a Superdex Peptide HR 10/30 column (Amersham-Pharmacia, Biotech, Uppsala, Sweden). Proteins and peptides were eluted with 0.05 % TFA, containing 5 % acetonitrile at a flow rate of 250 l/min, and monitored by absorbance at 214 nm. The peptide fraction was further separated by RP-HPLC on a Vydac C18 column (4.6 x 250 mm, particle size 5 m) with a linear acetonitrile gradient (10-50 %) for 1 h at a flow rate of 1 ml/min and 40 C. Peptides were detected at 214 nm. The chromatographic fractions were tested for the antifungal activity against Helminthosporium sativum and characterized by mass spectrometry (MS) and N-terminal sequencing. Mass spectra were acquired on a model Reflex III mass spectrometer (Bruker Daltonics, Bremen, Germany). Dehydrobenzoic acid was used as matrix. N-terminal amino acid sequences were determined by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems) according to the manufacturer's protocol.

Results and Discussion. Previous analysis showed that the seeds of T. kiharae contained several families of antimicrobial peptides with molecular masses from 3 to 6 kD. I n this work, we focused on the glycine-rich peptides. We discovered least eight glycine-rich peptides. Their molecular masses varied from 4,295 to 4,750 Da. N-terminal sequencing (from 10 to 50 amino acid residues) showed that they were homologous, extremely glycine-rich, and cysteine-free. In sequenced regions, glycine constituted 70-80 % of amino acids, and only few other amino-acid residues were found (Tyr, His, Pro, and Ala). A remarkable feature of these peptides was the presence of different repeat motifs, such as GnYPGH, GnYP or GnYPGR, where n is a variable number. Similar repeat motifs were discovered in glycine-rich proteins involved in plant defense against biotic and abiotic stress. The glycine-rich peptides are low-abundant constituents of T. kiharae seeds, their yields were as follows: 0.4 g/g dry weight for Tk-AMP-G1, 0.6 g/g for Tk-AMP-G2, 0.7 g/g for peptides Tk-AMP-G3 and Tk-AMP-G4. Preliminary antifungal tests with H. sativum showed that the Tk-AMP-G peptides caused morphological changes in the fungus at concentrations of 100-150 mg/ml in a dose-dependent manner. These data indicate that T. kiharae is a valuable source of glycine-rich peptides, whose biological activities will be investigated in more detail.

Reference.

• Garcia-Olmedo B, Molina A, Alamillo J, and Rodriguez-Palenzuela P. 1998. Plant defense peptides. Biopolymers (Peptide Science) 47:479-491.

 

The alterations in several yield-contributing traits in transgenic wheat. [p. 151-152]

T.V. Korostyleva, G.V. Kozlovskaya, and V.A. Pukhalskiy.

During the last years, knockout technology based on molecular mechanisms of silencing (RNA interference) is actively used in experiments to study of gene functions. Now this approach has become a tool for wheat research. The method includes transformation of plant with the genetic construct providing the synthesis or endogenous formation in the cells of double-stranded RNA homologous to any transcribed region of the target gene. The appearance in cells of such dsRNA triggers specific defensive mechanisms of restriction of dsRNA and mRNA with homology to it, which results in silencing of target gene. This process was believed to be very specific. However, recently it was reported that dsRNA might exert unspecific effects on expression of the genome and the genes of the transgenic construct.

Earlier in laboratory the transgenic plants of spring, common wheat cultivar Khakasskaya were obtained using Agrobacterium transformation in planta. T-DNA of these plants contains inverted repeat of 350-bp fragment of tetracycline resistance gene from pBR322 under the control of 35S CaMV promoter and nptII gene as marker. This construct provides the synthesis of untranslatable dsRNA in wheat cells. It is supposed that its nucleotide sequence has no homology to the wheat genome.

In the present study we have investigated morphological stability of 18 independent transgenic lines on T1 selfed progeny. The presence of T-DNA in individual plant has been confirmed by PCR-analyses with primer to inverted repeat sequence from pBR322. Segregation of T-DNA in progeny was observed only in 2 from 18 transgenic lines (with non -Mendelian ratio), in other lines all offspring plants had transgenic insertion. In contrast to T1 plants with normal phenotype, T2 progeny of some lines displayed alteration in several traits in field conditions. Using analysis of variance by statistical program AGROS 2.10 we have estimated a significance of differences between transgenic plants from individual lines and control group of non-transgenic wheat plants of Khakasskaya. The established changes manifested in the decrease of spike length (a significant decrease in 13 from 18 lines, i.e., 16.6 % of transformants), a spikelet number per spike (16.6 %), kernel weight (a significant decrease in 13 from 18 lines, i.e., 72 %), and in the increasing of productive tillering in wheat (11 %).

The reason of these changes might be in the shifts in the hormonal balance in transformants; however, this assumption demands additional investigations. We are studying inheritance of these alterations. With regard to the nature of observed changes, we can conclude that a high frequency of similar-type changes in independent lines cannot result of either insertion mutagenesis or somaclonal variability. We believe that the insertion expressing dsRNA induces unspecific pleiotropic effect on the recipient wheat genome.

 

 

 

N.I. VAVILOV RESEARCH INSTITUTE OF PLANT INDUSTRY
42, B. Morskaya Str., St. Petersburg, 190000, Russian Federation.

 

Genealogical analysis of winter bread wheat cultivars resistant and susceptible to Fusarium head blight from Southern Russia and Ukraine. [p. 152-156]

S.P. Martynov and T.V. Dobrotvorskaya.

We studied winter bread wheat cultivars from southern Russian Federation (Krasnodar, Stavropol, and Rostov regions) and the Ukraine. For the analysis, cultivars with known pedigrees and were used not conflicting with estimates of reaction to Fusarium spp. Among the lines were 40 resistant and moderately resistant cultivars (see Table 5) and 61 susceptible cultivars from southern Russia including Bezostaya 1 (Borojevic and Dencic 1988, Buerstmayr et al. 1996), Avrora (Borojevic and Dencic 1988), Lgovskaya 167 (Anonymous 1990), Bystritsa, Delta, Podarok Donu, Donskoj Mayak, Tarasovskaya Ostistaya, Zernogradka 10, Zarnitsa, Stanichnaya, Bat'ko, Donskoj Syurpriz, Zernogradka 11, Rodnik Tarasovskij (Anonymous 1994-2003), Sfera (Puchkov et al. 1996), Spartanka (Terekhina 1993), Dialog 1061-10, KN-1221-k-7-2-14, KN-201-90-k-2, KN-2503-h-112-6-11, KN-2503-h-112-6-3, KN-3071-h-16-16, KN-3303-h-117, KN-3385-h-756, KN-4594-h-370-41, KN-5835-h-427, Massiv 809-1016, Rannyaya 47, Zamena, Krasnodarskaya 70, Olimpiya 2, Skifyanka, Soratnitsa, Ejka, Novokubanka, Otrada, Yugtina, Krasnodarskaya 90, Nika Kubani, Ofeliya, Polovchanka, Azau, Aliza, Zimorodok, Nak, Umanka, Kupava, Uskoryanka, Krasota, Selyanka, Krasnodarskaya 99, Yashkulyanka, Fisht (Puchkov et al. 2001); and from Ukraine: Albatros Odesskij, Donetskaya 46 (Anonymous 1989), Kiyanka (Anonymous 1981), Lutescens 7 (Anonymous 1990), Mironovskaya Yubilejnaya (Mesterhazy 2001), Odesskaya 132 (Anonymous 1976-1995), and Odesskaya Polukarlikovaya (Babayants et al. 2001).

For each of cultivars a genetic profile was constructed and transfer of scab resistance from ancestors to the descendants was traced on genealogical trees with the help of the Genetic Resources Information and Analysis System GRIS3.5 (Martynov and Dobrotvorskaya 2000). All cultivars of the analyzed set are the descendants of the most popular cultivars Bezostaya 1 (frequency of presence in pedigrees 94.5 %), Mironovskaya 808 (62.6 %), and/or Odesskaya 16 (65.9 %). To reveal differences in the contributions of these parents in groups of resistant and susceptible cultivars the two-way ANOVA of the parentage coefficients for the randomized design was used (Table 1). The investigated factors were cultivar groups (factor A) with two gradations (resistant and susceptible) and dominant ancestors (factor B) with number of gradations b = 3.

Table 1. Two-way ANOVA of the parentage coefficients of the three most important ancestors of winter bread wheat cultivars from southern Russian Federation and the Ukraine (original data are transformed through arcsinx) . Items with an * are significant at P < 0.001.

Source	SS	DF	MS	F
General	40,936.9	202
Cultivar groups (factor A)	545.6	1	545.6	4.08*
Ancestors (factor B)	12,132.7	2	6,066.3	45.41*
Interaction (A x B)	1,940.3	2	970.1	7.26*
Error	26,318.3	197	133.6

The ANOVA (Table 1) has shown the significance of differences of groups of resistance/susceptibility (factor A) and ancestor contributions (factor B). The greatest interest represents interaction of the investigated factors. The significant interaction (A´B) specifies differences in distribution of the contributions of the same ancestors in groups of resistant and susceptible cultivars. The comparison of average coefficients of parentage in cultivar groups (Table 2) shows, that the contributions of cultivars Bezostaya 1 and Mironovskaya 808 are identical in both groups, and the contribution of Odesskaya 16 is significant higher in group of resistant cultivars. It is known, that Bezostaya 1 is susceptible to FHB (Borojevic and Dencic 1988, Buerstmayr et al. 1996), and the estimates of reaction to Fusarium spp. for Mironovskaya 808 are conflicting, from moderately resistant (Javor et al. 1997) up to susceptible (Buerstmayr et al. 1996). The results of our analysis show that most likely Mironovskaya 808, as well as Bezostaya 1, does not carry genes of resistance to FHB. Moderately resistant cultivar Odesskaya 16 (Kazmin and Shindin 1997) most frequently was the donor of resistance of scab-resistance genes in cultivars from southern Russian Federation and the Ukraine. Odesskaya 16 was selected from cultivar Odesskaya 12 (Hostianum 237/Zemka, where Hostianum 237 is selection from Kharkovskaya, a landrace of Kharkov region, and Zemka is local variety from Odessa region). Odesskaya 16 is absent in pedigrees of some resistant cultivars (Dakha, Russa, Echo, and Zimdar 4), but there are other derived cultivars of Hostianum 237. Therefore, we assume that a source of resistance to FHB in cultivars from southern Russian Federation and the Ukraine was the landrace Kharkovskaya via Hostianum 237.

Table 2. Average coefficients of parentage of important ancestors in groups of resistant to scab and susceptible winter bread wheat cultivars from southern Russian Federation and the Ukraine. Items with an * are significantly different at P < 0.01.

Ancestor name	Resistant	Susceptible
Bezostaya 1	0.37	0.38
Mironovskaya 808	0.15	0.16
Odesskaya 16	0.25*	0.10

To understand the role of other sources of resistance, we used ANOVA of the contributions of some known donors of resistance, which are in pedigrees of the investigated cultivar set (Table 3). The investigated factors were groups of resistant/susceptible cultivars (factor A) and donors of resistance genes (factor B) with the number of gradation b = 9. The ANOVA (Table 3) shows significant differences in the average parentage coefficients of groups of resistant/susceptible cultivars (factor A) and donors (factor B). The significant 'A x B' interaction specifies differences in distribution of the contributions of the donors in groups of resistant and susceptible cultivars.

Table 3. The two-way ANOVA of the parentage coefficients of donors of resistance to scab in pedigrees of bread wheat cultivars from Southern Russia and Ukraine (original data are transformed through arcsinx). Values with an * are significant at P < 0.001.

Source	SS	DF	MS	F
General	23578.6	246
Groups (factor A)	1669.8	1	1669.8	39.73*
Donors of resistance (factor B)	10716.3	8	1339.5	31.88*
Interaction (AxB)	1568.8	8	196.1	4.67*
Error	9623.7	229	42.0

Comparing average coefficients of parentage of the donors (Table 4) shows that in addition to Odesskaya 16 in the group of resistant cultivars are the significant cultivars Redcoat, Frontana, and Cheyenne. These donor cultivars are in pedigrees of the southern Russian and Ukrainian cultivars via Biserka (Redcoat), Red River 68 (Frontana), and Colt (Cheyenne).

Table 4. Average coefficients of parentage of the donors of scab-resistance genes in sets of resistant and susceptible winter wheat cultivars from southern Russian Federation and the Ukraine. Values with an * are significantly different at P < 0.05.

Ancestor name	Resistant	Susceptible
Odesskaya 16	0.25*	0.10
Redcoat	0.09*	0.03
Hope	0.01	0.00
Frontana	0.07*	0.03
Tohoku 34	0.01	0.01
Kooperatorka	0.03	0.02
Cheyenne	0.08*	0.01
Canus	0.01	0.01
Gentil Rosso	0.02	0.02

Established with the help of the pedigree analysis, the probable donors of resistance genes are given in Table 5. Of the 43 resistant and moderately resistant lines, three of the pedigrees are unknown and two cultivars (Maslovchanka 90 and Kharkovskaya 20) were not established. Most frequently, Odesskaya 16 and its derivatives (Odesskaya 51, Obrij) (55 %) and Frontana (28 %) were donors of resistance genes. Resistance to FHB in the older cultivars Kooperatorka, Novokrymka 102 (Ukraine), and Cheyenne (USA) were selected from the landrace Crimean, which probably was heterogenous for reaction to Fusarium spp. The Canadian cultivar Canus (Marquis/Kanred) also most likely received resistance genes from Crimean via Kanred, because Marquis is susceptible to scab.

This analysis has shown that despite of the rather large number of known sources of resistance to FHB in the Russian and Ukrainian wheat breeding programs, they are practically unused. Our analysis was made on the basis of the information about resistance or susceptibility of bread winter wheat cultivars received by the different authors in different time. Therefore, we consider the data about source of resistance and statistical estimations made by comparison of sets of resistant and susceptible cultivars, as preliminary. Nevertheless, the approach, based on the genealogical information, can be useful for the analysis of the comparable data.

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