GrainGenes

ITEMS FROM THE RUSSIAN FEDERATION (CONTINUED)

 

SIBERIAN INSTITUTE OF PLANT PHYSIOLOGY AND BIOCHEMISTRY
Ler montov str., 132, 664033, Irkutsk-33, P.O. Box 1243, Russian Federation.

 

Regulation of plant uncoupling protein CSP 310 activity in the shoots winter wheat seedlings during cold stress. [p. 156-158]

A.V. Kolesnichenko, V.V. Zykova, and V.K. Voinikov.

Recently, we found that in cereals such as winter wheat and winter rye, the cold stress protein CSP 310 caused uncoupling of oxidation and phosphorylation in mitochondria during cold stress (Kolesnichenko et al. 1996, Voinikov et al. 1998, Kolesnichenko et al. 1999). During cold stress, this cytoplasmic protein rapidly increases in amount (Kolesnichenko et al. 1996) and associates with mitochondria (Kolesnichenko et al. 2000a). On the other hand, this protein was present in nonstressed 3-day-old winter wheat shoots but did not cause uncoupling of oxidation and phosphorylation in mitochondria of nonstressed plants (Kolesnichenko et al. 2000b, Kolesnichenko et al. 2000c). The aim of this work was to study regulation of the CSP 310 uncoupling activity in 3-day-old winter wheat shoots during cold stress.

Materials and methods. Three-day-old etiolated shoots of winter wheat cultivar Irkutskaya Ozimaya) were grown on moist paper at 26°C. The cold-stress treatment was performed as describe in (Kolesnichenko et al. 2000c) at -1 C for 1 h. Antiserum against the stress protein CSP 310 was obtained as described previously (Kolesnichenko et al. 1996). Electrophoresis of native proteins was performed as described previously (Kolesnichenko et al. 2000d). The relative molecular weights of the proteins were determined by using a HMW kit of markers (Pharmacia, Sweden). Western blotting and staining of the gel by ethidium bromide were as described previously (Kolesnichenko et al. 2000c).

Results and discussion. The cold-stress treatment caused some changes in the spectrum of CSP 310-like winter wheat proteins (Fig. 1). In nonstressed shoots, cytoplasmic CSP 310-like proteins with molecular weights of 310 and 230 kDa, a number of proteins with molecular weights about 140 kDa, and CSP 310 subunits with molecular weights of 66 and 56 kDa were detected. In the spectrum of CSP 310-like proteins of stressed winter wheat shoots, only proteins with a molecular weight of 310 kDa was detected (Fig. 1). These data show that during cold stress some changes in the spectrum of CSP 310 occurs. We propose that during cold stress an association of CSP 310 subunits with LMW CSP 310-like proteins, which did not have uncoupling activity, into CSP 310 that could cause uncoupling of oxidation and phosphorylation in winter wheat mitochondria during cold stress.

Previously, cold stress in winter rye was shown to cause changes in features of CSP 310 (Kolesnichenko et al. 2000c). If nonstressed, CSP 310 was bound with nuclear acids (constituently synthesized form). In stressed plants, this protein was not bound with nuclear acids (stress-induced form). These two forms of CSP 310 differ in their uncoupling activity (Pobezhimova et al. 2001). If the constituently synthesized form did not cause significant uncoupling in the mitochondria, then the stress-induced form of CSP 310 had a high uncoupling activity.

The influence of cold stress on this feature of winter wheat shoots was investigated by using ethidium bromide gel coloring and gave the same picture as in winter rye (Fig. 2). If winter wheat shoots were nonstressed, this stress protein was bound with nuclear acid, in stressed shoots this protein was not bound with nuclear acid (Fig. 2). We concluded that a mechanism of regulation of CSP 310 uncoupling activity also exists in winter wheat.

Based on the data obtained, we propose that the mechanism that regulates the CSP 310 uncoupling activity in winter wheat consist of three parts. The first increases CSP 310 concentration because of its synthesis de novo. The second transforms the nonactive LMW CSP 310-like proteins into active CSP 310. The third part transitions the constituently synthesized form with low uncoupling activity into a stress-induced form with high uncoupling activity. We are suggesting that this mechanism allows winter wheat mitochondria to rapidly transform into a low-energy state and turn on plant mitochondrial thermogenic mechanisms.

Acknowledgments. The work has been performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko AV, Ostroumova EA, Zykova VV, and Voinikov VK. 1999. The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmic proteins of winter rye, winter wheat, Elymus and maize. J Therm Biol 24: 211-215.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000a. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Kolesnichenko AV, Zykova VV, Grabelnych OI, Sumina ON, Pobezhimova TP, and Voinikov VK. 2000b. Screening of mitochondrial proteins in winter rye, winter wheat, Elymus and maize with an immunochemical affinity to the stress protein 310 kD and their intramitochondrial localization in winter wheat. J Therm Biol 25:245-249.
• Kolesnichenko A, Zykova VV, and Voinikov V. 2000c. A comparison of the immunochemical affinity of cytoplasmic, mitochondrial and nuclear proteins of winter rye (Secale cereale L.) to a 310 kD stress protein in control plants and during exposure to cold stress. J Therm Biol 25:203-209.
• Kolesnichenko AV, Ostroumova EA, Zykova VV, and Voinikov VK. 2000d. Proteins immunologically related to a 310 kD stress protein in four grass species. Russ J Plant Physiol 47(2):176-179.
• Pobezhimova TP, Grabelnych OI, Kolesnichenko AV, and Voinikov VK. 2001. The comparison of uncoupling activity of constituently synthesized and stress-induced forms of winter rye stress uncoupling protein CSP 310. J Therm Biol 26:95-101.
• Voinikov V, Pobezhimova T, Kolesnichenko A, Varakina N, and Borovskii G. 1998. Stress protein 310 kD affects the energetic activity of plant mitochondria under hypothermia. J Therm Biol 23:1-4.

 

The difference between temperatures of hardened and nonhardened winter wheat seedling shoots during cold stress. [p. 158-159]

A.V. Kolesnichenko, O.I. Grabelnych, V.V. Tourchaninova, N.A. Koroleva, T.P. Pobezhimova, A.M. Korzun, and V.K. Voinikov.

Because of the particularities of an organism, plants were believed to be incapable of adjusting their temperature. However, in the1960s, during the flowering of Aroide, the strong activation of an alternative cyanide-resistant respiration pathway that accompanied thermogenesis was found (Wilson and Smith 1971). Some researches suggested that a cyanide-resistant alternative oxidase also can participate in the processes of plant thermoregulation during low-temperature stress (Vanlerberghe and McIntosh 1992). Recently, uncoupling proteins, which are homologues of mammalian mitochondrial uncoupling proteins (UCPs), were found to exist in plants (Vercesi et al. 1995). They were proposed to participate in plant protection from low-temperature stress (Laloi et al. 1997). Several years ago, the cytoplasmic protein CSP 310 was discovered. CSP 310 also causes uncoupling oxidation and phosphorylation in the mitochondria of winter cereals during low-temperature stress (Kolesnichenko et al. 1996). The mechanism of the CSP 310 uncoupling action is still unknown, but there are some data that show that CSP 310 is present in winter wheat mitochondria (Kolesnichenko et al. 2000). Living, nonhardened winter wheat shoots under cold shock (-4 C, 1 h) were shown to be able to generate heat and maintain a temperature above 0 C for the initial 25-30 min (Vojnikov et al. 1984), but it was not known if this process occurred in hardened plants. Therefore, the present work investigated the difference between temperature of nonhardened and hardened shoots of winter wheat during cold stress.

The temperature of chilled seedlings was recorded by a copper constant thermocouple with a sensitivity of approximately 0.025°C (wire diameter 0.1 mm) connected to the input of a highly sensitive microvoltmeter. For measuring, seedling shoots (3 g) were tightly packed in a small container at 20 C and transferred to the thermostat with an experimental temperature (0 or -4 C). Temperature changes were recorded for 1 h. The shoot samples were then placed in hot water (95 C) to stop all metabolic processes, and temperature changes were recorded as the killed samples cooled. Thus, we obtained temperature curves for living and dead tissue with one tissue sample following chilling and calculated the temperature difference between killed and living seedling shoot tissue.

When we studied the temperature difference between living and killed nonhardened (control) winter wheat shoots at different temperatures of cold stress, we found no significant difference between the maximum of these values at 0 and -4 C (Figs. 3 and 4). Both temperatures were about 2.5 C after 15 min of cold stress. At the same time, in experiments with winter wheat at 0 C, we observed an increase in the temperature difference between living and killed shoots from 0.5 C at 30 min to 1 C at 50-55 min of cold stress (Fig. 3). We did not observe such changes during experiments at -4 C (Fig. 4). In this case, the temperature difference between the living and killed shoots decreased to less than 0.5°C after 30 min of cold stress and did not increase later.

An analysis of the influence of hardening (4 C, 7 days) on the temperature difference between living and killed winter wheat shoots showed that hardening significantly changed this value both at 0 and -4 C (Figs. 3 and 4). In hardened winter wheat seedlings shoots both at 0 and -4 C, the maximum temperature difference between living and killed shoots during first 15 min of cold stress was about 0.5 C (Figs. 3 and 4). The temperature difference then was reduced to approximately 0 C for 15-30 min of cold shock. But after 45 min, the temperature differences increased to 0.5 C and after 50-60 min of cold stress, the difference between rows of hardened living and nonhardened living shoots disappeared (Figs. 3 and 4).

In our opinion, the presence of thermogenic systems in cold-resistant winter cereal seedlings can be connected with the life cycle. Ice formation inside cells will kill plants (Levitt 1980). Cereals can withstand autumn frosts during the short time that the temperature is below 0 C. Winter cereals have a number of defense systems that allow them sufficiently pump water from protoplasm to apoplast, and, thereby, avoid ice formation inside their cells. Plant cells have many other defense systems connected with the synthesis of different classes of stress proteins such as dehidrins (Kolesnichenko et al. 2000b). However, some time is necessary for activations of these systems in nonhardened plants. We propose that a fast uncoupling of oxidation and phosphorylation in winter wheat mitochondria and thermogenesis caused by this process allow seedlings some time for activation of these systems. On the other hand, for hardened plants it is not necessary to use energy for heating because all defense systems are activate by this time. We suggest that the thermogenesis systems in the winter cereals are an adjustment to the winter life-style and protection from frosts and overcooling in nonhardened plants.

Acknowledgments. The work was performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko A, Zykova V, and Voinikov V. 2000a. A comparison of the immunochemical affinity of cytoplasmic, mitochondrial and nuclear proteins of winter rye (Secale cereale L.) to a 310 kD stress protein in control plants and during exposure to cold stress. J Therm Biol 25:203-209.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000b. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Laloi M, Klein M, Riesmeier JW, Muller-Rober B, Fleury C, Bouilland F, and Ricquier D. 1997. A plant cold-induced uncoupling protein. Nature 389:135-136.
• Levitt J. 1980. Chilling injury and resistance. In: Chilling, freezing and high temperature stresses. Responses of plant to environmental stresses (Kozlowsky TT ed). Academic Press, New York. I:23-64.
• Vanlerberghe GC and McIntosh L. 1992. Lower grows temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol 100:115-119.
• Vercesi AE, Martins IS, Silva MAP, Leite HMF, Cuccovia IM, and Chaimovich H. 1995. PUMPing plants. Nature 375:24.
• Vojnikov V, Korzun A, Pobezhimova T, and Varakina N. 1984. Effect of cold shock on the mitochondrial activity and on the temperature of winter wheat seedlings. Biochem Physiol Pflanzen 179:327-330.
• Wilson RH and Smith BH. 1971. Uncoupling of Sauromatum spadix mitochondria as a mechanism of thermogenesis. Z Pflanzenphysiol 65:124-129.

 

The influence of known plant uncoupling protein function on lipid peroxidation in winter wheat seedlings shoots during cold stress. [p. 160-163]

A.V. Kolesnichenko, O.I. Grabelnych.,V.V. Zykova, Y.Y. Tourchaninova, N.A. Koroleva, T.P. Pobezhimova, and V.K. Voinikov.

Injury to chilling and freezing causes symptoms in plants that frequently are coincident with peroxidation of free fatty acids (Parkin et al. 1989). Peroxide and malondialdehyde levels often are increased by freezing and thawing, suggesting that the peroxidation of lipids (Loubaresse et al. 1991) results in structural and functional membrane and membrane proteins changes (Shewfelt and Erickson 1991). Mitochondria are one of the major sources of superoxide, a powerful oxidant radical, in chilling-sensitive plant tissues at low temperatures (Purvis et al. 1995). Even in nonstress conditions, 1-2 % of the oxygen reduced in mitochondria by iron-sulfur centers in complex I and partially by reduced ubiquinone and cytochrome b in complex III is constitutively converted to superoxide (Richter et al. 1995, Skulachev 1998). Mitochondria also are a major site for the accumulation of LMW Fe2+ complexes, which induce lipid peroxidation in membranes (Tangeras et al. 1980, Minotti and Aust 1987).

At the same time, cold stress caused increased levels of antioxidants in many plant species. Furthermore, different isoenzymes of glutathione reductase also are expressed during cold acclimation of red spruce (Picea rubens) needles. One of these isoenzymes is proposed to be a cold-acclimation protein (Hausladen and Alschler 1993).

Recently, it was suggested that in mitochondria there is another physiological mechanism involved in the antioxidant defense system of the cell. Skulachev proposed that the uncoupling of oxidation and phosphorylation in mitochondria at the conditions of oxidation stress could decrease ROS formation by mitochondrial respiratory chain generation (Skulachev 1994). This point of view was supported by recent studies of plant UCP-like uncoupling proteins such as PUMP and others. For example, the inhibition of PUMP activity in isolated potato tuber mitochondria was found to significantly increase mitochondrial H2O2 generation. Substrates of UCP-like uncoupling proteins such as Linoleic acid and other free fatty acids also reduce mitochondrial H2O2 generation (Korshunov et al. 1999, Kowaltowski et al. 1999).

We know that PUMP and other UCP-like proteins are not the only uncoupling systems in plant mitochondria. All plants have in the mitochondria mechanism such as the alternative cyanide-resistant oxidase (AOX) that is activated during cold stress and causes a decrease of oxidation and phosphorylation (Vanlerberghe and McIntosh 1992). Recently, cereals, such as winter wheat and winter rye, were found to contain the cold-stress protein CSP 310 (Kolesnichenko et al. 1996, Kolesnichenko et al. 1999), which also causes uncoupling of oxidation and phosphorylation in mitochondria during cold stress (Voinikov et al. 1998). The addition of some concentrations of CSP 310 to isolated winter wheat mitochondria induced ascorbate-dependent and NADH-dependent lipid peroxidation systems (Zykova et al. 2000). However, the reactions of isolated organelles and whole plant cell on the same treatment can be different.

The aim of this study is to examine the influence of activation and inhibition of different plant uncoupling systems on lipid peroxidation in the shoots of winter wheat seedling during cold stress.

Materials and methods. Three-day-old etiolated shoots of the winter wheat cultivar Irkutskaya Ozimaya were grown on moist paper at 26 C. The shoot samples (3 g) were infiltrated with 1, water; 2, pyruvate (40 mM); 3, BHAM (40 mM); 4, linoleic acid (40 mM); 5, prokaine (40 mM); 6, CSP 310 (1 mg/ml); or 7, anti-CSP 310 antiserum (2 mg/ml) for 1 h and were packed in paper containers for stress treatment. A cold stress of the shoots was given in a thermostat at -4 C for 1 h. The control plants were not subjected to the stress conditions. Stress protein CSP 310 and the antiserum against this protein were obtained as described previously (Kolesnichenko et al. 1996).

The rate of lipid peroxidation was determened by measuring the primary products of lipid peroxidation-conjugated diene formation as described previously (Zykova et al. 2000) with some modifications. The sample of seedling shoots (3 g) after treatment was homogenized with 6 ml of media containing 175 mM KCl and 25 mM Tris-HCl (pH 7.4). To measure the dienic conjugate content, plant cell lipids were extracted with a hexane/isopropanol (1:1 v/v) mixture (9 ml/1 ml of homogenate) by shaking. After shaking, 1 ml of H2O was added to stratify the hexane and isopropanol phases. Measurements of the dienic conjugate content were made in the mixture of hexane phase (0.5 ml) with C2H5OH (3 ml) at 233 nm on a spectrophotometer SF-46 (LOMO, USSR). The dienic conjugate content of the sample was calculated according to 233-nm M extinction coefficient to polyunsaturated fatty acids conjugated dienes 2.2 x 10^5^/M/sm (Recknagel and Ghoshal 1966).

The oxygen uptake of winter wheat shoots was analyzed polarographically at 27 C using a platinum electrode of a closed type in a 1.4-ml volume cell. The reaction mixture contained 50 mM KH2PO4 and 50 mM sucrose (pH 5.2).

All experiments were repeated six times. The data obtained were analyzed statistically and arithmetic means and standard errors were determined.

Results and discussion. The cold stress treatment caused approximately a 20 % increase of dienic conjugate formation in winter wheat shoot tissue (Fig. 5). These data correlate well with the data obtained of other investigators about the influence of cold stress on lipid peroxidation in plant tissues (Parkin et al. 1989, Loubaresse et al. 1991). We also observed a slight increase of oxygen consumption in winter wheat shoots during cold stress (Fig. 6).

The infiltration of winter wheat shoots by all the investigated activators of plant uncoupling mitochondrial systems (Pyruvate, which activates AOX; Linoleic acid, which is the substrate of PUMP and activates it; and CSP 310) reduced the rate of lipid peroxidation during cold stress (Fig. 5). Of interest is the fact that all of them caused approximately a 30 % decrease in the formation of dienic conjugates, and these values were slightly lower but were not statistically differed from the value of dienic conjugate content in nonstressed winter wheat shoots (Fig. 5). These activators influence oxygen uptake in winter wheat shoots during cold stress show by causing an increase of oxygen uptake (Fig. 6). If infiltration of the shoots by pyruvate and CSP 310 caused a 20-25 % increase of oxygen consumption, their infiltration by linoleic acid caused an approximately 100 % increase in oxygen consumption. These data allows us to propose that infiltration of winter wheat shoots by linoleic acid can strongly activate UCP-like plant uncoupling proteins.

At the same time, inhibitors of plant uncoupling mitochondrial systems (BHAM, which inhibits AOX; Procaine, which is the inhibitor of phospholipase A2 and, therefore, reduces the amount of free fatty acids that are substrates of PUMP; and anti-CSP 310 antiserum, which inhibits uncoupling activity of CSP 310 in winter wheat mitochondria) differ in their influence on lipid peroxidation in winter wheat shoots (Fig. 5). If infiltration of winter wheat shoots by BHAM caused a significant (~ 30 %) increase of lipid peroxidation, an infiltration of shoots by procaine and anti-CSP 310 antiserum did not cause an increase in the formation of dienic conjugates (Fig. 5). Unlike their influence on lipid peroxidation, all these inhibitors caused approximately a 25 % decrease of oxygen consumption by winter wheat shoots (Fig. 6)

Interesting data were obtained on the influence of linoleic acid and procaine on the dienic conjugate amounts in winter wheat shoots during cold stress. Because linoleic acid is one of substrates of lipid peroxidation, one can suppose that the infiltration of winter wheat shoots would cause an increase in the formation of dienic conjugates. Our data show that under conditions of physiological stress, an increase in linoleic acid concentration in the plant cell causes a decrease in the formation of lipid peroxidation products, and the effect is similar to those observed in isolated mitochondria (Korshunov et al. 1999, Kowaltowski et al. 1999). At the same time, a decrease in the free fatty-acids content due to infiltration of winter wheat shoots by procaine because of phospholipase A2 inhibition did not have any significant influence on the rate of lipid peroxidation during cold stress. We propose that it takes place because of two processes; an increase of ROS formation by mitochondria because of inhibition of PUMP activity and a decrease in free fatty-acid content, which are substrates of lipid peroxidation.

Based on these data, we conclude that during cold stress all studied winter wheat uncoupling systems participate in plant defense against oxidative stress.

Acknowledgments. This work was performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Hausladen A and Alscher RG. 1993. Glutathione In: Antioxidants in Higher Plants (Alscher RG and Hess JL eds). CRC Press, Boca Raton, FL. pp. 1-30.
• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko AV, Ostroumova EA, Zykova VV, and Voinikov VK. 1999. The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmic proteins of winter rye, winter wheat, Elymus and maize. J Therm Biol 24:211-215.
• Kolesnichenko A, Zykova V, and Voinikov V. 2000a. A comparison of the immunochemical affinity of cytoplasmic, mitochondrial and nuclear proteins of winter rye (Secale cereale L.) to a 310 kD stress protein in control plants and during exposure to cold stress. J Therm Biol 25:203-209.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000b. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Korshunov SS., Korkina OV, Ruuge EK, Skulachev VP, and Starkov AA. 1999. Fatty acids as natural uncouplers preventing generation of O2 and H2O2 by mitochondria in the resting state. FEBS Letters 435:215-218.
• Kowaltowski AJ, Costa ADT, and Vercesi AE. 1999. Activation of the potato plant uncoupling mitochondrial protein inhibits reactive oxygen species generation by the respiratory chain. FEBS Letters 425:213-216.
• Loubaresse M, Paulin A, and Dereuddre J. 1991. Effects of freezing on membrane lipid peroxidation of rhododendron roots (Rhododendron cv. Demontague, Jean-Marie). Compte Rendu Acad Sci 313:453-459.
• Minotti G and Aust SD. 1987. An investigation into the mechanism of citrate-Fe2+-dependent lipid peroxidation. Free Radic Biol & Med 3:379-387.
• Parkin KL, Marangoni A, Jackman R, Yada R, and Stanley D. 1989. Chilling injury. A review of possible mechanisms. J Food Biochem 13:127-153.
• Purvis AC, Shewfelt RL, and Georgenie JW. 1995. Superoxide production by mitochondria isolated from green bell pepper fruit. Physiol Plant 94:743-749.
• Recknagel RO and Ghoshal AK. 1966. Lipoperoxidation of rat liver microsomal lipids by carbon tetrachloride. Nature 210:1162-1163.
• Richter Ch, Gogvadze V, Laffranchi R, and Schlapbach R. 1995. Oxidants in mitohondria: from physiology to diseases. Biochem Biophys Acta 1271:67-74.
• Shewfelt RL and Erickson ME. 1991. Role of lipid peroxidation in the mechanism of membrane-associated disorders in edible plant tissue. Trends Food Sci Technol 2:152-154.
• Skulachev VP. 1994. Lowering of the intracellular O2 concentration as a special function of respiratory systems of the cells. Biochemistry (Moskow) 59:1910-1912.
• Skulachev VP. 1998. Possible role of reactive oxygen species in the antiviral defense. Biochemistry (Moskow) 63:1691-1694.
• Tangeras A, Flatmark T, Bäcktröm D, and Ehrenberg A. 1980. Mitochondrial iron content not bound in heme and iron-sulfur centers. Biochim Biophys Acta 589:162-175.
• Vanlerberghe GC and McIntosh L. 1992. Lower grows temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol 100:115-119.
• Voinikov V, Pobezhimova T, Kolesnichenko A, Varakina N, and Borovskii G. 1998. Stress protein 310 kD affects the energetic activity of plant mitochondria under hypothermia. J Therm Biol 23:1-4.
• Zykova VV, Grabelnych OI, Antipina AI, Koroleva NA, Vladimirova SV, Kolesnichenko AV, Pobezhimova TP, and Voinikov VK. 2000. Plant stress-related uncoupling protein CSP 310 caused lipid peroxidation in winter wheat mitochondria under chilling stress. J Therm Biol 25:323-327.

 

The influence of antisera toward some cytoplasmic proteins on the energetic activity of mitochondria in the shoots of wheat seedlings. [p. 163-164]

O.I. Grabelnych, A.V. Kolesnichenko, S.I. Misharin, A.I. Antipina, T.P. Pobezhimova, and V.K. Voinikov.

Cold stress causes some changes in the content of different stress proteins in plants (Kolesnichenko et al. 2000a). The study of the content of different antigens in cold-resistant and cold-sensitive winter wheat cultivars showed that there is a significant difference between them in the content of the two antigens with Rf 0.30 and 0.24. Cold-resistant winter wheat cultivars contained higher amounts of antigen with Rf 0.30 and lower amounts of antigen with Rf 0.24 than the cold-sensitive cultivars (Misharin et al. 1997). Cold stress caused a significant increase in the amount of antigen with Rf 0.30 and a decrease of antigen with Rf 0.24 (Misharin et al. 1997). Further investigations showed that antigen with Rf 0.30 has the native molecular weight 310 kDa and consists of two types of subunits with molecular weights of 56 and 66 kDa (Kolesnichenko et al. 1996, Voinikov et al. 1998). This protein is a cold-shock protein with a molecular weight of 310 kDa (CSP 310) (Voinikov et al. 1998). One of the functions of CSP 310 during cold stress is an uncoupling of oxidative phosphorylation in mitochondria during cold stress (Voinikov et al. 1998). This uncoupling takes place because CSP 310 associates with mitochondria during cold stress (Kolesnichenko et al. 2000b). The study of changes in content of the antigens Rf 0.30 (CSP 310) and with Rf 0.24 during cold stress (Misharin et al. 1997) led us to study their influence on energetic activity of winter wheat mitochondria. To study this, antisera against CSP 310 and the antigen Rf 0.24 were used.

Materials and methods. Three-day-old etiolated shoots of winter wheat were grown on moist paper at 26 C. Antisera against CSP 310 and an antigen with Rf 0.24 were obtained as described previously (Kolesnichenko et al. 1996).

Mitochondria were isolated from the shoots of winter wheat seedlings by differential centrifugation (Pobezhimova et al. 1996). The energetic activity of winter wheat mitochondria was analyzed polarographically at 27°C using a closed-type platinum electrode in a 1.4 ml cell (Estabrook 1967). The reaction mixture contained 125 mM KCl, 18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. The oxidation substrate was 10 mM Malate in the presence of 10 mM glutamate.

All the experiments were made in six separate preparations. The data obtained were analyzed statistically, i.e. arithmetic means and standard errors were determined.

Results and discussion. Antisera against CSP 310 and the antigen with Rf 0.24 were added to a polarograph cell to the mitochondria of winter wheat at state 4. The data obtained showed that antisera against CSP 310 and the antigen with Rf 0.24 had an antagonistic influence on the energetic activity of incubated mitochondria (Fig. 7). An addition of anti-CSP 310 antiserum to incubated mitochondria at state 4 caused a decrease in nonphosphorylative respiration (Fig. 7A) and an increase in the respiratory control (RC) value (Fig. 7B). These changes increased the coupling of oxidation and phosphorylation processes. The coupling effect of anti-CSP 310 antiserum increased with increased amounts of antiserum added up to the concentration of antiserum added (2.4 mg/ml of mitochondria incubation medium). The addition of higher concentrations of antiserum did not cause further decreases in nonphosphorylative respiration (Fig. 7A) or increases in the RC value (Fig. 7B).

On the other hand, the addition of antiserum against the antigen with Rf 0.24 caused an increase of nonphosphorylative respiration (Fig. 7A) and a decrease of RC coefficient (Fig. 7B). However, the influence of an antiserum against the antigen with Rf 0.24 was not as strong as that of the anti-CSP 310 antiserum.

Previously, an addition to incubated mitochondria stress protein CSP 310 was shown to cause an increase of nonphosphorylative respiration and a decrease in the RC value (Voinikov et al. 1998). An addition to incubated mitochondria antiserum against CSP 310 caused the reverse effect; a decrease of the nonphosphorylative respiration and an increase in the RC value. Based on this data, we propose that the addition to incubated mitochondria isolated antigen with Rf 0.24 would have a coupling effect on mitochondria. Previous work has shown that CSP 310 and the antigen with Rf 0.24 in winter wheat are antagonists; the higher the content of CSP 310 during autumn and winter, the higher the content of the antigen with Rf 0.24 in winter wheat in the spring (Misharin et al. unpublished data). On the basis of these data, we suggest that these two proteins (CSP 310 and the antigen with Rf 0.24) participate in the regulation of the energetic activity of winter wheat mitochondria not only during cold stress but also during different studies of ontogenesis.

Acknowledgments. This work was performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Estabrook RW. 1967. Mitochondrial respiratory control and the polarografic measurement of ADP:O ratio. Methods Enzymology 10:41-47.
• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko AV, Pobezhimova TP, and Voinikov VK. 2000a. Cold-shock proteins in plants. Russ J Plant Physiol 47:549-554.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000b. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Misharin SI, Antipina AI, and Voinikov VK. 1997. The cold shock effect on antigen composition of winter rye and wheat. Physiol Biochem Cultiv Plants (Ukr) 29:215-219.
• Pobezhimova TP, Voinikov VK, and Varakina NN. 1996. Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vitro by elevated temperature. J Therm Biol 21:283-288.
• Voinikov V, Pobezhimova T, Kolesnichenko A, Varakina N, and Borovskii G. 1998. Stress protein 310 kD affects the energetic activity of plant mitochondria under hypothermia. J Therm Biol 23:1-4.

 

The influence of cold stress on the peroxidation of lipids in the respiratory chain in the mitochondria of different winter wheats. [p. 164-165]

V.V. Zykova, A.V. Kolesnichenko, O.I. Grabelnych, V.V. Tourchaninova, and V.K. Voinikov.

In plants, the development of symptoms from chilling injury is frequently coincident with the peroxidation of fatty acids (Parkin et al. 1989). The source of activated oxygen during freezing stress has not been established exactly, but experimental evidence indicates that mitochondria are a major source of superoxide in chilling-sensitive plant tissues at low temperatures (Purvis et al. 1995). About 1-2 % of the oxygen reduced in mitochondria by iron-sulfur centers in complex I and partially by reduced ubiquinone and cytochrome b in complex III is constitutively converted to superoxide, which is a powerful oxidant radical. However, these data were mainly obtained with the use mammalian mitochondria (Chakraborti et al. 1999). Therefore, the present work was to investigate the influence of cold stress on the peroxidation of lipids in the mitochondria of winter wheat during the functioning of different respiratory chain complexes.

The rate of lipid peroxidation was determined by measuring the primary products of lipid peroxidation conjugated during diene formation. Different substrates were used to determine the peroxidation of lipid in different mitochondrial respiratory chain complexes. Malate was used to study complex I, succinate to complex II, NADH to complex III, and ascorbate+TMPD to complex IV.

The data obtained showed that if mitochondria were isolated from nonstressed shoots of winter wheat, electron transfer occurred through complexes I, II, or III; and the rate of lipid peroxidation was equal and rather low (Fig. 8). In complex IV, the rate of lipid peroxidation was approximately 50 % higher (Fig. 8).

An explanation of these results is the fact that in the first complex function, electrons are transferred through an ubiquinone complex, which in plants can function as an effective antioxidant system because of alternative oxidase activity (Pobezhimova and Voinikov, 2000). In different mitochondrial respiratory chain complexes, different rates of lipid peroxidation induction were detected. The highest rate of dienic conjugates formation was detected in complex IV.

The influence of low-temperature stress on the rate of lipid peroxidation in the function of different respiratory chain complexes in mitochondria isolated from wheat shoots at 4 C did not caused an increase of dienic conjugates formation in any of the respiratory chain complexes. The data showed that if electron transfer was through complex I, the rate of lipid peroxidation in nonstressed and stressed shoots was rather low and equal (Fig. 8). The electrons transfer through complexes II, III, and IV, and the rate of lipid peroxidation in stressed plants were even lower, then in non-stressed (Fig. 8). We suppose that this fact occurs because of activation of antioxidant defense systems during cold stress.

Thus, we conclude that in winter wheat mitochondria, unlike mammals, the highest lipid peroxidation was detected in complex IV. Cold stress did not cause an increase of lipid peroxidation in the functioning of any mitochondrial respiratory chain complexes.

Acknowledgments. The work was performed, in part, with the support of the Russian Foundation of Basic Research (project 0004-48093).

References.

• Chakraborti T, Das S, Mondal M, Roychoudhury S, and Chakraborti S. 1999. Oxidant, mitochondria and calcium: an overview. Cell Signal 11:77-85.
• Parkin KL, Marangoni A, Jackman R, Yada R, and Stanley D. 1989. Chilling injury. A review of possible mechanisms. Food Biochem 13:127-153
• Pobezhimova TP and Voinikov VK. 2000. Biochemical and Physiological aspects of ubiquinone function. Membr Cell Biol 13:1-8.
• Purvis AC, Shewfelt RL, and Gegogeine JW. 1995. Superoxide production by mitochondria isolated from green bell pepper fruit. Physiol Plant 94:743-749.

 

The function of stress protein CSP 310 in winter wheat mitochondria. [p. 166-167]

O.I. Grabelnych, T.P. Pobezhimova, A.V. Kolesnichenko, and V.V. Voinikov.

The cold stress protein CSP 310, previously isolated from stressed shoots in winter rye, was found in winter wheat mitochondria (Kolesnichenko et al. 2000a). In vitro CSP 310 caused uncoupling of oxidation and phosphorylation (Voinkov et al. 1998), because of its association with mitochondria (Kolesnichenko et al. 2000b). CSP 310 and proteins immunochemically related to CSP 310 were found in the protein spectrum of winter wheat mitochondria (Kolesnichenko et al. 2000a). This present work studied the influence of cold-shock, stress-protein CSP 310 and anti-CSP 310 antiserum on the energetic activity of winter wheat mitochondria.

Mitochondria were extracted from the shoots of winter wheat by differential centrifugation as describes previously (Pobezhimova et al. 1996). The activity of mitochondria was recorded polarografically at 27 C using a platinum electrode of a closed type in a 1.4 ml volume cell. The data obtained were analyzed statistically and arithmetic means and standard errors were determined.

The study of the influence of cold shock (-l C, 1 h) on the energetic activity of winter wheat mitochondria showed that a cold shock caused significant uncoupling of oxidation and phosphorylation. In mitochondria isolated from nonstressed shoots, the rate of nonphosphorylative (state 4) respiration was 24.8±1.7 nMol O2/mg of mitochondrial protein and respiratory control coefficient (RC) was 3.97±0.09. In mitochondria isolated from stressed shoots these values were 41.7±1.4 nMol O2/mg of mitochondrial protein and 2.56±0.09, accordingly.

The influence of CSP 310 (0.5 mg/mg of mitochondria protein) on the energetic activity of mitochondria isolated from winter wheat showed that the addition of this protein to mitochondria incubated for 590 min at 0 C in vitro caused the uncoupling effect. This uncoupling effect was detected after the first 5 min of mitochondria incubation and characterized by a 40 % increase in state-4 respiration, as compared to the control mitochondria (see Fig. 9). Uncoupling of oxidation and phosphorylation in the mitochondria amount to about 60 % with 30 60 min incubation and remained on this level in the 90 min incubation (see Fig. 9). We now can propose that during cold shock in winter wheat mitochondria, uncoupling can be caused by the association of CSP 310 with mitochondria in vivo. To show the uncoupling effect of CSP 310 in vivo, an experiment with the addition of anti-CSP 310 antiserum to isolated mitochondria winter wheat was done.

To study the influence of anti-CSP 310 antiserum on the mitochondrial energetic activity, mitochondria were isolated and incubated for 30 min with and without addition of anti-CSP 310 antiserum. We saw a significant coupling of oxidation and phosphorylation. The rate of state-4 respiration decreased from 23.8±1.1 nMol O2/mg of mitochondrial protein in the control mitochondria to 12.1±0.6 nMol O2/mg of mitochondrial protein in mitochondria after anti-CSP 310 treatment. The RC coefficient after treatment increased up to 6.73±0.34. The nonimmune antiserum failed to cause any changes in the energetic activity of the mitochondria.

Based on these data, we concluded that the presence of the cold-shock protein CSP 310 is connected with the uncoupling of oxidation and phoshorylation mechanism in winter wheat mitochondria and is an adaptive reaction of plant to low-temperature stress.

Acknowledgments. The work has been performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Kolesnichenko AV, Zykova VV, Grabelnych OI, Sumina ON, Pobezhimova TP, and Voinikov VK. 2000a. Screening of mitochondrial proteins in winter rye, winter wheat, Elymus and maize with immunochemical affinity to the stress protein 310 kD and their intramitochondrial localization in winter wheat. J Thermal Biol 25(3):245-249.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000b. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Pobezhimova TP, Voinikov VK, and Varakina NN. 1996. Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vi tro by elevated temperature. J Thermal Biol 21:283-288.
• Voinikov V, Pobezhimova T, Kolesnichenko A, Varakina N, and Borovskii G. 1998. Stress protein 310 kD affects the energetic activity of plant mitochondria under hypothermia. J Thermal Biol 23:1-4.
  

 

An immunochemically related influence to plant stress uncoupling protein CSP 310 proteins in some cereal species on the energetic activity and lipid peroxidation of mitochondria in vitro. [p. 167-169]

V.V. Zykova, O.I. Grabelnych, N.A. Koroleva, T.P. Pobezhimova, Yu.M. Konstantinov, A.V. Kolesnichenko, and
V.K. Voinikov.

The plant cold-stress protein CSP 310 was isolated from winter rye (Kolesnichenko et al. 1996) and was found to have uncoupling activity (Voinikov et al. 1998). The association of CSP 310 with winter wheat mitochondria has been shown (Kolesnichenko et al. 2000a). At the same time, the presence of a family of immunochemically related to CSP 310 proteins in cereals was established. Proteins immunochemically related to CSP 310 are present in the cytoplasm (Kolesnichenko et al. 1999) and mitochondria (Kolesnichenko et al. 2000b) of cereals with different cold tolerance. By using affinity chromatography in a column with immobilized BrCN-activated Sepharose, anti-CSP 310 antiserum preparations immunochemically related to CSP 310 cytoplasmic proteins from winter rye, winter wheat, maize, and Elymus were obtained. These preparations consisted of 470, 310, 230, 132, 122, 112, 66, and 56 kDa proteins in preparations from winter rye; 230, 132, 122, 112, 66, and 56 kDa in winter wheat and maize; and 380-330, 230, 132, 122, 112, 66, and 56 kDa in Elymus (Kolesnichenko et al. 1999).

The aim of our current work is to examine an influence of the proteins immunochemically related to CSP 310 on energetic activity and lipid peroxidation in winter wheat mitochondria in vitro during cold stress.

Crude and purified mitochondria were isolated from winter wheat shoots using a discontinuous Percoll gradient consisting of 18, 23, and 40 % Percoll (Voinikov et al. 1998). In these experiments, 0.5 mg of proteins immunochemically related to CSP 310 were added to a mitochondrial suspension per 1 mg of mitochondrial protein. The activity of the mitochondria was recorded polarographically immediately after isolation and after 30 min of incubation. Malate in the presence of 10 mM glutamate was used as an oxidation substrate. Polarograms were used to calculate the respiratory control by Chance-Williams (Estabrook 1967).

The rate of lipid peroxidation was determined by measuring the primary products of lipid peroxidation conjugated diene formation. For induction of the ascorbate-dependent lipid peroxidation system, 1 mM ascorbate, 1 mM ADP, and 20 µM Fe+2 were added to the incubation media. For induction of the NADH dependent lipid peroxidation system, 1 mM NADH, 1 mM ADP, and 20 mkMol Fe2+ were added to the incubation media. The data obtained were analyzed statistically; the level of statistical significance was P < 0.05 (n = 6).

The influence of CSP 310-like proteins of winter rye, winter wheat, Elymus, and maize on the functional activity of winter wheat mitochondria during hypothermia in vitro is shown in Fig. 10. The incubation of mitochondria for 30 min at 0 C with CSP 310-like proteins from winter rye caused a decrease in respiratory control. The addition of CSP 310-like proteins from maize to incubated mitochondria caused only a slight decrease in respiratory control. At the same time, the addition of CSP 310-like proteins from winter wheat and Elymus to mitochondria failed to result in any significant changes in their activity. Based on these data, we concluded that only CSP 310 and no other composition of its subunits caused uncoupling oxidation and phosphorylation in mitochondria.

Previously, we showed that the addition of exogenous CSP 310 to winter wheat mitochondria caused an increase in lipid peroxidation; conjugated diene formation (Zykova et al. 2000). The influence of CSP 310-like cytoplasmic proteins from the species studied on lipid peroxidation shows that CSP 310-like proteins from winter wheat, maize, and Elymus do not have the peroxiding effect as from winter rye (Fig. 11). Proteins from Elymus even have an antioxidant activity in all systems studied (Fig. 11). We propose that one function of CSP 310 is the disassembling of a part of the damage in cold-stressed mitochondria by activation of lipid peroxidation followed by apoptosis (Skulachev 1994). In this case, nonactive LMW combinations of CSP 310 subunits can be a 'depot' that allows for a rapid increase in CSP 310 concentration during cold stress.

Acknowledgments. The work was performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Estabrook RW. 1967. Mitochondrial respiratory control and the polarografic measurement of ADP:O ratio. Method Enzymol 10:41-47.
• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko AV, Ostroumova EA, Zykova VV, and Voinikov VK. 1999. The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmatic proteins of winter rye, winter wheat, Elymus and maize. J Therm Biol 24:211-215.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000a. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Kolesnichenko A, Zykova V, Grabelnych O, Sumina O, Pobezhimova T, and Voinikov V. 2000b. Screening of mitochondrial proteins in winter rye, winter wheat, Elymus and maize with an immunochemical affinity to the stress protein 310 kD and their intramitochondrial localisation in winter wheat. J Therm Biol 25:245-249.
• Skulachev VP. 1994. Lowering of the intracellular O2 concentration as a special function of respiratory systems of the cells. Biochemistry (Moskow). 59:1910-1912.
• Voinikov V, Pobezhimova T, Kolesnichenko A, Varakina N, and Borovskii G. 1998. Stress protein 310 kD affects the energetic activity of plant mitochondria under hypothermia. J Therm Biol 23: 1-4.
• Zykova VV, Grabelnych OI, Antipina AI, Koroleva NA, Vladimirova SV, Pobezhimova TP, Kolesnichenko AV, and Voinikov VK. 2000. Plant stress-related uncoupling protein CSP 310 caused lipid peroxidation in winter wheat mitochondria under chilling stress. J Therm Biol 25:323-327.

 

The influence of different plant thermogenic systems on heat generation in shoots of winter wheat seedlings during cold stress. [p. 169-170]

A.V. Kolesnichenko, O.I. Grabelnych, V.V. Tourchaninova, N.A. Koroleva, T.P. Pobezhimova, A.M. Korzun, V.V. Zykova, and V.K. Voinikov.

The herbaceous winter cultivars of cereals such as wheat and rye are very frost resistant and can tolerate winter temperatures less than -15 to -20 C. Winter cereals have many different physiological and biochemical mechanisms that allow them to survive at such low temperatures. Many of these mechanisms, now well studied, include changes in lipid and phospholipid metabolism (Smolenska and Kuiper 1977, Nishida and Murata 1996, Murata and Los 1997), increases in unsaturated fatty acids amounts (De Santis et al. 1999), and increases in sugar content (Levitt 1980). These processes enable the plant to avoid intracellular ice formation and stabilize and protect cell membranes (Murata and Los 1997). All the changes in a plant cell take place during the first step of hardening in the autumn, which takes about 1-2 weeks (Levitt 1980). At the same time, in autumn, winter cereals outlive autumn frosts when temperatures fall very rapidly.

Previously, we showed that under cold shock (-4 C, 1 h) living shoots of winter wheat can generate heat and maintain a temperature above 0 C for the initial 25-30 min (Vojnikov et al. 1984). At the same time, it was found that only a part of this difference was detected in KCN-infiltrated shoots (Vojuikov et al. 1984). Many have suggested that the heat liberated during cold shock in winter wheat shoots might be produced because of energy transfer from oxidative processes in mitochondria. Until recently, uncoupling proteins, which are homologues of mammalian mitochondrial uncoupling proteins (UCPs), were thought to exist in plants (Vercesi et al. 1995, Laloi et al. 1997). On the other hand, the cytoplasmic protein CSP 310 was discovered some years ago, which also uncouples oxidation and phosphorylation in winter cereals mitochondria during low-temperature stress (Kolesnichenko et al. 1996). The mechanism of the CSP 310 uncoupling action is still unknown, but there are some data show that it is differ then fatty acid cycling mechanism of known UCP-like uncoupling mitochondrial proteins. The present work is devoted to the study of heat generation by different thermogenic systems (alternative cyanide-resistant oxidase, UCP-like proteins, and CSP 310) in plants during cold stress.

The temperature of chilled seedlings was recorded by a copper-constantan thermocouple with a sensitivity of approximately 0.0-25 C. (wire diameter 0.1 mm) connected to the input of a high-sensitive microvoltmeter. For measurements, shoots of seedlings (3 g) were tightly packed in a small container at 20 C and then transferred to a thermostat with the experimental temperature (0 or -40 C). Temperature changes were recorded for 1 h. The shoot sample was then placed in hot water (95 C) to stop all metabolic processes. Temperature changes were recorded in the killed samples cooled from 20 C to the experimental temperature. Thus, we obtained temperature curves following chilling with one tissue sample for both living and dead tissue and calculated the temperature difference (DT) between killed and living shoot tissue in seedlings.

The analysis of the influence of different plant thermogenic systems on temperature difference between living and killed winter wheat shoots was at -4 C. During the experiments, we found that infiltration of shoots by KCN, which inhibits the transport of electrons on the main pathway of the respiratory chain and functions only in alternative cyanide-resistant oxidase, caused a decrease in the temperature difference between the KCN-treated living and killed winter wheat shoots in the first 15-20 minutes of cold shock from 2 to 1 C (Fig. 12). When the temperature difference was reduced to near 0 C after 30-50 min of cold shock, temperature differences between raw KCN-treated living and living shoots disappeared after 60 min (Fig. 12).

The infiltration of winter wheat shoots by procaine, which inhibits phospholipase activity and decreases the quantity of free fatty acids (Scherphor et al. 1972), also reduced heat generation of winter wheat shoots. The decrease in the temperature difference between the procaine-infiltrated living and killed shoots of winter wheat in the first 15-20 min of cold shock from 2 to 1.5 C was marked in this case (Fig. 12). Thereafter, the temperature difference between procaine-infiltrated living and KCN-infiltrated living decreased. After 30 min of cold shock, the temperature difference between procaine-infiltrated living and KCN-infiltrated living disappeared (Fig. 12).

The infiltration of winter wheat shoots by the antiserum against stress protein CSP 310, and the infiltration of shoots by procaine, caused a decrease in the difference between anti-CSP 310 infiltrated living and killed winter wheat shoots during the first 15-20 min of cold shock at 2 to 1.5 C (Fig. 12). However, after the temperature of the anti-CSP 310-infiltrated living and the control living shoots decreased and 30 min of cold shock, the difference between them disappeared (Fig. 12).

We can assume that there are at least three thermogenic systems in winter wheat that allow survival during cold shock.

Acknowledgments. The work has been performed, in part, with the support of the Russian Foundation of Basic Research (project 00-04-48093).

References.

• Kolesnichenko AV, Borovskii GB, Voinikov VK, Misharin SI, and Antipina AI. 1996. Antigen from winter rye accumulated under low temperature. Russ J Plant Physiol 43:771-776.
• Kolesnichenko A, Zykova V, and Voinikov V. 2000a. A comparison of the immunochemical affinity of cytoplasmic, mitochondrial and nuclear proteins of winter rye (Secale cereale L.) to a 310 kD stress protein in control plants and during exposure to cold stress. J Therm Biol 25:203-209.
• Kolesnichenko A, Grabelnych O, Pobezhimova T, and Voinikov V. 2000b. The association of plant stress uncoupling protein CSP 310 with winter wheat mitochondria in vitro during exposure to low temperature. J Plant Physiol 156:805-807.
• Laloi M, Klein M, Riesmeier JW, Muller-Rober B, Fleury C, Bouilland F, and Ricquier D. 1997. A plant cold-induced uncoupling protein. Nature 389:135- 136.
• Levitt J. 1980. Chilling injury and resistance. In: Chilling, Freezing and High Temperature Stresses. Responses of Plant to environmental Stresses, V. 1 (Kozlowsky TT ed). Academic Press, NY, pp. 23-64.
• Vanlerberghe GC and McIntosh L. 1992. Lower grows temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol 100:115-119.
• Vercesi AE, Martins IS, Silva MAP, Leite HMF, Cuccovia IM, and Chaimovich H. 1995. PUMPing plants. Nature 375:24.
• Vojoikov V, Korzun A, Pobezhimova T, and Varakina N. 1984. Effect of cold shock on the mitochondrial activity and on the temperature of winter wheat seedlings. Biochem Physiol Pflanzen 179:327-330.
• Wilson RH and Smith BH. 1971. Uncoupling of Sauromatum spadix mitochondria as a mechanism of thermogenesis. Z Pflanzenphysiol 65:124

 

Dehydrin-like proteins in the wheat mitochondria after freezing, drought, and ABA treatment. [p. 170-172]

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

Near-freezing and freezing temperatures of drought stress can induce cellular dehydration when water from within the cell migrates to outside the cell (Guy 1990). At the cellular level, plants respond to freezing and dehydration stress by the synthesis of specific stress proteins. The dehydrin family of proteins is induced by both cold and drought stress. Dehydrins, also referred to as Group II late embryogenesis abundant (LEA) proteins, are glycine-rich, hydrophilic, and thermostable. Dehydrins have been hypothesized to function by stabilizing large-scale hydrophobic interactions such as membranes structures or hydrophobic patches of proteins (Close 1996, Close 1997). An acidic dehydrin has been determined to localize in close proximity to the plasma membrane during cold acclimation supporting the role of cryoprotection of the plasma membrane during dehydration and freezing stress (Danyluk et al. 1998). Dehydrins stabilize other proteins or associated membranes because of its propensity to adopt alpha-helical structures in the presence of sodium dodecyl sulfate and the apparent polypeptide adhesion properties (Ismail et al. 1999). Immunolocalization and subcellular fractionation results have established that members of the dehydrin family localize to the nucleus, cytoplasm, and plasma membrane. Recently, we found that two dehydrin-like proteins (dlps) accumulate in mitochondria of T. aestivum), S. cereale, and in Z. mays in response to cold stress (Borovskii et al. 2000). We also demonstrated a positive correlation between the accumulation of both mitochondrial dlps and interspecific variation of cold tolerance in response to cold treatment.

The objective of this study was to determine whether dlps localize to wheat mitochondria in response to stimuli other than those for cold adaptation. Here we report that drought, freezing stress, and exogenous ABA treatment also result in accumulation of dlps in the wheat mitochondria.

Materials and methods. Three-day-old etiolated seedlings of the winter wheat cultivar Irkutskaya Ozimaya were grown at 23 C. Unstressed plants were maintained under growth conditions for 1 day. A freezing stress was given at -10 C for 20-30 min until ice crystallization on the surface of the seedlings. Two-day-old seedlings transferred to dry filter paper for 1 day at the growing conditions served as a drought stress. ABA treatments were made at the control temperature by spraying of a 1 mM ABA solution containing 0.1 % Tween-20. ABA-treated seedlings were harvested the day after treatment. Control and treated seedling were compared at similar growth stages.

Crude mitochondria isolation and purification was performed as described previously (Borovskii et al. 2000). Purified mitochondria were dissolved in the sample loading buffer for SDS-PAGE and boiled for 3 min. Proteins were subjected to SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad, USA) according to manufacturer's instructions. Western blotting and immunodetection were as described earlier (Borovskii et al. 2000). A primary antibody (1:1,000 dilution) raised against dehydrin was kindly provided by Dr. T.J. Close. All experiments were replicated three to four times.

Results and discussion. Five dlps were found in the mitochondria of winter wheat (Fig. 13). The bands corresponding to all these proteins were very weak or lacking if antibodies blocked by dehydrin peptides were used (data not shown). Two of these polypeptides (63 and 52 kDa) were thermostable (Fig. 14). Three other proteins did not seem to be thermostable; 58, 56, and 28 kDa (Fig. 13). Finding proteins immunologically related to dehydrins but unstable to high temperature is unusual but such a situation does occur. Li and coworkers (1998) describe constitutive and not thermostable proteins related to dehydrins in fucoid algae. Of more importance is that nonthermostable wheat mitochondrial dlps were not induced by all the treatments used (Fig. 13). Based on this observation, we concluded that these proteins were not involved in the stress reaction and adaptation.

Freezing stress caused a high accumulation of dlp63 in the wheat mitochondria (Fig. 13, lane 2), whereas dlp52 was not registered under freezing stress. During adaptation to cold, this polypeptide also was accumulated (Borovskii et al. 2000) (Fig. 13, lane 2). Drought stress did little to raise of the content of dlp63 (Fig. 13, lane 3), but there was a more clear increase in dlp52 (Fig. 13, lane 3) although cold adaptation caused a large accumulation of this protein (Borovskii et al. 2000. Both proteins (dlp52 and dlp63) were induced sharply by ABA treatment (Fig. 13, lane 4).

The data obtained revealed that the cold-inducible mitochondrial dlp63 and dlp52 may have different regulation in winter wheat. The dlp63 was induced by both short exposure to freezing temperatures and ABA treatment, whereas dlp52 was induced only by ABA. An ABA-associated response to low temperature was elucidated by a slow increase in ABA content during cold treatment. Low temperatures lead to a 3- to 4-fold increase in ABA content. Drought stress leads to more than a 20-fold increase in ABA level during first day of exposure (Lang et al. 1994). Synthesis of dlp63 ppears to have at least two induction phases; the first can be induced very rapidly in ABA-independent manner under freezing and the second is slower in the case of ABA-dependence cold and drought. Dlp52 seems to be induced only in ABA-dependent manner and may account for the fact that dlp52 is strongly associated with wheat mitochondria under 1-day drought and ABA treatment, less associated during cold acclimation, but not associated under freezing stress.

The accumulation of dehydrins during cold acclimation has been studied in different plant species (Close 1996, Sarhan et al. 1997). In wheat and other cereals, dehydrins are coordinately regulated by low temperatures and accumulated in high levels in freezing tolerant members of Poaceae. Our results concerning the accumulation of dehydrin-like proteins in mitochondria under the cold treatment and drought and freezing stress indicate that mitochondrial dlps might be involved in freezing- and dehydrative-tolerance mechanisms. The difference in quantity and rapidity of increasing of stress-inducible dlps possibly reflects the stability of mitochondria, cells, and the whole plant to dehydration and freezing stress. Further experiments concerning dlp localization in mitochondria are in progress.

Acknowledgments. The research was funded by the Russian Foundation of Basic Research (project 99-04-48121). We sincerely thank Dr. T.J. Close for gift of the dehydrin antibody and dehydrin-specific peptide.

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.
• 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 commonality 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.
• Guy CL. 1990. Cold acclimation and freezing tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41:187-223.
• 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.
• Lang V, Mantila E, Welin B, Sundberg B, and Palva ET. 1994. Alterations in water status, endogenous abscisic acid content, and expression of rab18 gene during the development of freezing tolerance in Arabidopsis thaliana. Plant Physiol 104:1341-1349.
• Li R, Brawley SH, and Close TJ. 1998. Proteins immunologically related to dehydrins in fucoud algae. J Phycol 34:642-650.
• 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.

 

Cold and ABA-inducible proteins of winter wheat. [p. 172-174]

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

Because the freezing tolerance of winter wheat is inducible, the induction is commonly assumed to involve the synthesis of novel cold-regulated peptides (COR) that confer freezing tolerance to the tissue by means of their enzymatic activity or structural properties.

Many of the COR-genes that encode the COR-proteins have been isolated and characterized from a variety of plant species (Thomashow 1998). Most of these genes are responsive to abscisic acid. Increasing freezing tolerance is provided by ABA-independent and ABA-dependent pathways that partly interact and converge to amplify the induction of specific cold-responsive genes (Ishitani et al. 1997, Madhani and Fink 1997). Although many of the genes that are induced during cold acclimation encode COR-proteins with known activities, many do not. Interestingly, most of these have a set of distinctive properties in common: they are unusually hydrophilic; many remain soluble upon boiling in dilute aqueous buffer; many have relatively simple amino acid compositions, and being composed largely of a few amino acids; many are composed largely of repeated amino acid motifs (Thomashow 1999).

The proportion of the heat stable COR-proteins during cold adaptation is about 10 % of total cell protein fraction (Guy 1990). Because of this reason, visualization of COR-proteins in the total protein spectra is difficult. In this connection our study was devoted to heat stable COR-proteins. The aims of the present work were: (a) to find COR-proteins, characteristic of hardened winter wheat; (b) to study cold induction of these polypeptides; (c) to compare these COR-proteins with ABA-inducible peptides.

Materials and methods. Three-day-old etiolated seedlings of T. aestivum were grown at 22 C. Unstressed plants were maintained under growth conditions for one day else. A mild cold treatment (acclimation) was given by subjecting seedlings to a temperature of 4 C for 9 days. Control and cold-treated seedlings were compared at similar growth stages. Water-soluble proteins were extracted from seedlings. Plant material (3.5 g) frozen in liquid nitrogen was ground to a fine powder with a mortar and pestle. Proteins were extracted with 100 mM Tris-HCl, pH 7.6, containing 10 mM dithiothreitol, 10 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The homogenate obtained was centrifuged at 17,000 x g for 15 min using the angle rotor of a K-24 centrifuge (Janetzki, Germany). To isolate heat-stable proteins, the supernatant was boiled for 20 min in a water bath and then centrifuged at 17,000 x g for 15 min to remove insoluble proteins (Lin et al. 1990). Total and heat-stable proteins were precipitated with 10 % trichloroacetic acid and separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978). Proteins were subjected to polyacrylamide gel electrophoresis with sodium dodecyl sulfate SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad, USA) according to manufacturer's instruction. For each lane, 15 µg of protein were loaded.

ABA was applied exogenously. Seedlings were exposed to 1,000 µM ABA with the addition of Tween-20 (0.1 % solution) during 1 day. In order to study changes in heat-stable protein synthesis during cold and ABA exposure, proteins were labeled in vivo with 14C-leucine, separated by SDS-gel electrophoresis. Radioactive proteins were visualized by fluorography. All experiments were replicated three times.

Results and discussion. Several specific heat-stable proteins (209, 196, 169, 66, 50, and 41 kDa) were found in wheat seedlings hardened for 9 days at 4 C (Fig. 15, lanes 1 and 2). These proteins accumulated gradually in the process of hardening over the course of 9 days. A correlation between the gradual hardening of winter wheat seedlings and the accumulation of heat stable proteins implies the importance of these proteins in low temperature tolerance of plants. These results are in agreement with the data of other authors (Lin et al. 1990, Karasev et al. 1991, Thomashow 1999). Some of the heat stable proteins (209, 196, and 66 kDa) were synthesized at a high rate in response to chilling, whereas the synthesis of other proteins (169 and 41 kDa) was only slightly enhanced. Thus, protein accumulation during the hardening of winter wheat seedlings does not always result from the induction of their syntheses. Some proteins that accumulated in the cold also are synthesized in unhardened seedlings (Fig. 16).

COR-polypeptides of cold adapted cereals with similar molecular weights were detected by Canadian researchers (Houde et al. 1995). These proteins belong to the WCS 120 family (wheat cold specific), which is characteristic of frost-resistant cereals and includes five polypeptides with molecular weights of 200, 180, 66, 50, and 40 kDa related to dehydrins. The proteins of dehydrin family have cryoprotective property. The content of the 50- and 66-kDa proteins were considerably higher than those of other WCS proteins, which agrees with our results (Fig. 15, lanes 1 and 2). The differences in molecular weight of our proteins and peptides detected by the Canadian researchers can be ascribed to the low accuracy of estimating molecular weight in electrophoretic studies. We also supposed cryoprotective properties of revealed wheat COR-proteins.

To order compare these COR-proteins with ABA-inducible peptides of wheat, an ABA exposition was used. ABA treatment produced more proteins than low temperature acclimation (Fig. 15, Fig. 16, and Fig. 17). Whereas ABA-treated seedlings of wheat accumulated 11 ABA-inducible proteins with molecular weights of 228, 209, 196, 97, 66, 56, 50, 48, 46, 41, and 32 kDa (Fig. 15), cold-adapted plants accumulated only six with molecular weights of 209, 196, 169, 66, 50, and 41 kDa. Thus, it appears that all COR-proteins characteristic of hardened state of winter wheat with molecular weights of 209, 196, 169, 66, 50, and 41 kDa are induced by cold and ABA. Synthesis of protein 169 kDa is activated only by cold.

Acknowledgments. The work has been performed, in part, with the support of the Russian Foundation of Basic Research (project 99-04-48121).

References.

• Esen AA. 1978. A simple method for quantitative, semiquantitative and qualitative assay of protein. Ann Biochem 89:264-273.
• Guy CL. 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41:187-223.
• 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.
• Ishitani M, Xiong L, Stevenson B, and Zhu J-K. 1997. Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 9:1935-1949.
• Karasev GS, Narleva GI, Boruakh KK, and Trunova TI. 1991. Alterations in the composition and content of polypeptides during acclimation of winter wheat to below-zero temperatures. Fiziol Biokhim Kult Rast 23:480-485.
• Lin C, Guo WW, Everson E, and Thomashow MR. 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol 94:1078-1083.
• Madhani HD and Fink GR. 1997. Combinatorial control required for the specificity of yeast MARK signaling. Science 275:1314-1317.
• Thomashow MF. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann Rev Plant Physiol Plant Mol Biol 50:571-599.
• Thomashow MF. 1998. Role of cold-responsive genes in plant freezing tolerance. 118:1-7.

 

Accumulation and disappearance of heat-stable RABs depending on the freezing tolerance of winter wheat plants at different developmental phases. [p. 175-177]

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

Many plants have the capability to acclimatize to cold. Exposure to low but nonfreezing temperatures can increase the freezing tolerance of the plant substantially and is accompanied by changes in the hormonal balance toward accumulation of abscisic acid (Guy 1990). Analysis of the intracellular levels of ABA during cold acclimation shows a peak in ABA early in the acclimation process (Heino et al. 1990). This fact, together with the induction of freezing tolerance by exogenously added ABA, suggests that ABA is a one of trigger-factors that initiate the acclimation process. Indeed, a number of studies revealed ABA-dependent and ABA-independent stress signaling pathways that cross-talk and converge to activate stress COR-gene (Cold Regulated genes) expression.

Thus, during low temperature adaptation, the proteins responsive to ABA (RAB-proteins) and other ABA-uninducible polypeptides are synthesized. Some of the RAB proteins have are heat stable (Robertson et al. 1994). The heat-stable RAB polypeptides provide greater protection to heat-induced coagulation than the control heat-stable proteins. The heat stability of selected RABs may reflect the ability of these proteins to endure harsh environments such as extremes of pH or ionic strength (Leung and Giraudat 1998).

Some studies have established positive correlation between drought and cold-stress tolerance and the accumulation of heat-stable RABs in different plant species (Leung and Giraudat 1998). As far as we know, information about the accumulation and disappearance of the RABs under field conditions, where all complex of nature factors act, is almost lacking.

To this end, the aim of our present work is to find proteins with an immunochemical affinity to one of the RABs (RAB-16) among the heat-stable protein fraction of winter wheat crowns and to elucidate whether the these proteins accumulate and disappear relative to the winter tolerance of winter wheat plants at different developmental phases under field conditions. This project is of particular interest to us because the climate of eastern Siberia is severe. Especially dangerous is the period in the spring when frequent low freezing temperatures occur.

Materials and methods. Crowns of winter wheat cultivar Irkutskaia ozimaia were used in the study. This genotype is winter-resistant and highly productive under the severe climatic conditions of the eastern Siberia.

All experiments were planted in the field, and seeds were sown at different times: 15 and 25 August and 5 September. Crowns or etiolated, underground stem segments (depending on the phase of plant development) were sampled in the field, frozen in liquid nitrogen, and processed for protein extraction in the laboratory. All samples were collected every 15 days throughout the entire autumn (beginning in November) and spring growth cycles and, also, monthly in the winter.

For the region of eastern Siberia, early frosts are characteristic. After the snow thaw at the end of March/beginning of April, the daily air temperature varied from 12 to -17 C. During this time, the temperature of the soil surface could change by 40 C during the day (from a night temperature of -13 C to a daytime temperature of 27 C).

Winter hardiness in the plants was determined as the number of plants surviving in the spring. Three sites for plant counting, with a total area of 1 m2, were marked in the autumn on each experimental plot.

Water-soluble, heat-stable, and total proteins were extracted from the crown of the plants as previously described by Lin et al (1990) with modifications by Stupnikova et al. (1998). The total and heat-stable proteins were separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978).

Proteins were separated in SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad, USA) according to the manufacturer's instructions. Fifteen µg of protein were loaded in each lane. Western blotting and immunodetection were as described by Timmons and Dunbar (1990) using anti-RABs antibodies (1:1000 dilution), kindly provided by J. Mundy (Denmark, University of Copenhagen, pers commun). Western blot images were analyzed using the Sigma Scan Pro Software (Sigma Chemicals, USA). The relative content of proteins were estimated in the conventional units. All the experiments were done in three replicates. Figure 18 illustrates representative electrophoregrams. Table 1 lists the average numbers of surviving plants for 6 years (1990-96) and their standard errors.

 

 

Results and discussion. A large difference in overwintering between the winter wheat plants of cv. Irkutskaia ozimaia sown at different times was observed (15, 25 August and 5 September, see Table l). When plants were sown later, they produced only one or two tillers or were at the 3-leaf phase. Such plants survived much better than plants sown on 15 August.

The cold acclimation of winter wheat plants in the field resulted in the accumulation of several heat-stable proteins with molecular weights of 209, 196, 66, and 50 kDa (Fig. 18A). Western-blotting was with antibodies to RABs of heat-stable proteins extracted by SDS from winter wheat crowns. This technique revealed that COR-polypeptides with molecular weights of 209, 196, 66, and 50 kDa were related to RABs (Fig. 18B). At the same time, an in crease of another RAB with a molecular weight of 41 kDa (Fig. 18B) was observed. This RAB was not detected on electrophoregrams (Fig. 18A) because of its small content and high sensitivity of immunoblotting. This immunochemical affinity suggests that the COR-polypeptides first have protecting functions characteristic of the RAB-family, and, second, are apparently induced by ABA.

All winter wheat plants of different ages accumulated RABs during the autumn. The medium- and high-molecular part of protein spectrum of these plants did not change throughout entire winter and spring until April (Fig. 19A and B). However, the amount of RABs depended on the developmental phase. Before winter, the younger plants (sown on 5 September) accumulated less heat-stable COR-proteins than plants sown on 15 August. The morphologically more advanced plants (sown on 15 August) contained more RABs (Fig. 19A) and depended on gradual decreasing temperatures in the autumn. Biosynthesis of RABs was suppressed. Thus, winter wheat plants sown earlier developed at higher temperatures than ones sown later and accumulated more RAB-polypeptides.

Quantitative changes in RABs occurred in April after an increase in temperatures and snow thaw. By the middle of April (sampling on 16 April, 1996), differences between plants sown at different times was more distinct. By this time, the pattern of RABs was the opposite of that observed in the autumn. Plants sown earlier, which accumulated more of these proteins before the onset of winter, lost these proteins more rapidly in the spring and returned to the unhardened state. In contrast, the content of heat-stable RABs in younger plants was significantly higher than in morphologically more advanced plants (Fig. 19B). In May, no qualitative or quantitative differences were observed between plants sown at different times, except for 50-kDa protein. However, the differences in the amounts of this protein also were small (Fig. 19C).

The available data show a connection between winter hardiness and the content of heat-stable RABs in the crown during the spring (Table 1, Fig. 19B). Because the survival of winter wheat in eastern Siberia is determined by plant tolerance in the spring, the concentration of these proteins at this time seems to be vital. We propose that heat-stable RAB proteins may have 'salvation' properties and confer tolerance to freezing. Theoretically, the heat-stable RAB proteins characterized here could undergo noncovalent protein­protein or protein­lipid domain interactions with other proteins, macromolecular structures (ribosomes), or membranes to prevent heat- or stress-induced inactivation, denaturation, or coagulation.

References.

• Esen AA. 1978. A simple method for quantitative, semi-quantitative and qualitative assay of protein. Annals Biochem 89:264-273.
• Guy CL. 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41:187-223.
• Heino P, Sandman G, Lang V, Nordin K, and Palva ET. 1990. Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 79: 801-806
  Leung J and Giraudat J. 1998. Abscisic acid signal transduction. Ann Rev Plant Physiol Plant Mol Biol 49:199-222.
• Lin C, Guo WW, Everson E, and Thomashow MR. 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol 94:1078-1083.
• Robertson AJ, Ishikawa M, Gusta LV, and MacKenzie SL. 1994. Abscisic acid-induced heat tolerance in Bromus inermis Leyss cell-suspension cultures. Plant Physiol 105:181-190.
• Stupnikova IV, Borovskii GB, and Voinikov VK. 1998. Accumulation of hat-stable proteins in winter wheat seedlings during hypothermia. Rus J Plant Physiol 45:744-748.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.

 

Thermostable cold-regulated proteins in different wheat species after cold acclimation. [p. 178-179]

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

Low-temperatures activate protective biochemical systems that play a main role in acclimation of plants to cold temperatures. All of these processes are regulated at the level of gene expression and lead to the synthesis and accumulation of various stress proteins or COR-polypeptides (cold regulated). The majority of the described and identified COR-proteins are hydrophilic in nature, thermostable, and have a simple amino acid composition (Arora and Wisniewski 1994, Hughes and Dunn 1996, Uemura et al. 1996, Boothe et al. 1997, Thomashow 1999). Previously, we have shown the accumulation of thermostable COR-polypeptides under hardening of seedlings of some species of cereals. In general, the polypeptides of a high and average molecular weight for the stable species, and average and low molecular weight for the unstable species, are accumulated. For the freeze-stable cultivar of winter wheat Irkutskaia ozimaja, the mass of these proteins are 209, 196, 169, 66, and 50 kDa (Stupnikova et al. 1998). We are interested in how these polypeptides, characteristic for the stable plants, are represented in other members of Triticeae.

Materials and methods. Etiolated seedlings of T. aestivum, T. monococcum, T. durum, T. timopheevii, T. zhukovskyii, and T. spelta were used. Seedlings were grown on wet filter paper for 3 days at 22 C and acclimated for 7 days at 4 C. Water-soluble, heat-stable and total proteins were extracted from the seedlings as described previously (Lin et al. 1990) with modifications (Stupnikova et al. 1998). Total and heat-stable proteins were separated electrophoretically. The gels were analyzed by the Sigma Scan Pro Software (Sigma Chemical, USA). Relative protein contents were estimated in the conventional units. All the experiments were performed in three replicates. The figures illustrate representative electrophoregrams.

Results and discussion. Before acclimation to cold, no thermostable COR-proteins with molecular masses of 209, 196, 169, 66, and 50 kDa were observed in the SDS-PAGE profiles of the species studied (Fig. 20). The accumulation of these proteins in winter wheats after cold treatment were noted earlier (Stupnikova et al. 1998). No serious differences in the total protein between the wheat species of cold acclimated seedlings were seen in the SDS-PAGE spectra (data not shown). Noticeable differences could be demonstrated if the thermostable proteins of acclimated seedlings were compared (Fig. 21). Only one COR-protein with a molecular mass different from that previously noted was found; a 204 kDa in T. zhukovskyii (Fig. 21, lane 3). Other COR-proteins of the wheat species have the same molecular masses as in T. aestivum. The abundance of each COR-protein varied from species to species. For comparison, we scanned the gels and analyzed the total intensity of corresponding bands. The results were summarized in Table 2. COR-proteins are known to have an additive protective effect, thus, we also list the sum of the intensities of the all thermostable COR-proteins (Table 2).

 

 

The data suggest that each genome has its own thermostable COR-proteins that probably have an additive effect on freezing stability. Nevertheless, the A but mostly the D genomes give the main contribution to the diversity and abundance of thermostable COR-proteins in wheat. Triticum aestivum and T. spelta not only have the largest sum of all proteins intensities but also have the most different polypeptides (Table 2).

Acknowledgments. The research was funded by the Russian Foundation of Basic Research (project 99-04-48121).

References.

• Arora R and Wisniewski M. 1994. Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica (L.) Batsch.). II. A 60-kilodalton bark protein in cold-acclimated tissues of peach is heat stable and related to the dehydrin family of proteins. Plant Physiol 105:95-101.
• Boothe JG, Sonnichsen FD, de Beus MD, and Johnson-Flanagan AM. 1997. Purification, characterization, and structural analysis of a plant low-temperature-induced protein. Plant Physiol 113:367 -376.
• Dorofeev VF and Migushova EF. 1979. Wheat. Vol. 1. In: Flora of Cultivated Plants (Dorofeev VF and Korovina ON eds). Leningrad (St. Petersburg), Russia. Kolos (in Russian).
• Hughes MA and Dunn MA. 1996. The molecular biology of plant acclimation to low temperature. J Exp Bot 47:291-305.
• Lin C, Guo WW, Everson E, and Thomashow MR. 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol 94:1078-1083.
• Stupnikova IV, Borovskii GB, and Voinikov VK. 1998. Accumulation of heat-stable proteins in winter wheat seedlings during hypothermia. Russ J Plant Physiol 45:744-748.
• Thomashow MF. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571-599.
• Uemura M, Gilmour S, Thomashow M, and Steponkus P. 1996. Effects of COR6.6 and COR15am polypeptides encoded by COR (cold-regulated) genes of Arabidopsis thaliana on the freeze-induced fusion and leakage of liposomes. Plant Physiol 111:313-327.

 

The disappearance of heat-stable, dehydrin-related proteins and survival in winter wheat in the spring is dependent from the age of plants. [p. 179-185]

G.B. Borovskii, I.V. Stupnikova, A.A. Peshkova, N.V. Dorofeev, and V.K. Voinikov.

Plants respond to cold and freezing temperatures through physiological, morphological, and metabolic processes. A number of studies have demonstrated a role for osmoprotectants and changes in the membrane composition in cold-stress tolerance (Galinski 1993, Bohnert et al. 1995, Hare et al. 1998). Development of plant tolerance to low temperatures is accompanied by the synthesis of specific stress proteins that play an important role in this process (Guy 1990). Therefore, studying the biochemical and physiological processes underlying plant adaptation to low-temperature stress and the loss of frost tolerance during winter thaws and in the spring is important.

Genetic systems of the plant cell rapidly respond to low temperature by expression of new proteins (Guy 1990, Alberdi and Corcuera 1991, Hughes and Dunn 1996, Thomashow 1998). The synthesis of COR proteins (cold regulated) was repeatedly demonstrated in diverse plant species. In some cases, only a cold treatment induced the synthesis of COR proteins (Ouellet et al. 1993, Weretilnyk et al. 1993), whereas other COR proteins also were controlled by ABA, water deficiency, and salinity stress (Houde et al. 1992, Robertson et al. 1994, Yamaguchi-Shinozaki et al. 1994, Mantyla et al. 1995). The function of COR proteins is not yet elucidated. One class of proteins that is induced by both cold and drought stress are the dehydrin family of proteins. Dehydrins, also referred to as group II late embryogenesis abundant (LEA) proteins, are glycine-rich, hydrophilic, and thermostable. Dehydrins are conserved evolutionarily among photosynthetic organisms including angiosperms, gymnosperms, ferns, mosses, liverworts, algae, and cyanobacteria, and in some nonphotosynthetic organisms such as yeast (Close and Lammers 1993, Close 1996, Campbell and Close 1997, Mtwisha et al. 1998). A number of studies have established positive correlation between drought and cold stress tolerance and dehydrin accumulation in a number of different plant species and different genotypes of a species (Labhilili et al. 1995, Moons et al. 1995, Close 1996, Pelah et al. 1997). Dehydrins have been hypothesised to function by stabilizing large-scale hydrophobic interactions such as membrane structures or hydrophobic patches of proteins (Dure 1993; Close 1996, 1997). 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 during conditions of cellular dehydration or low temperatures. An acidic dehydrin has been determined to localize in close proximity to the plasma membrane during cold acclimation supporting the role of cryoprotection of the plasma membrane during dehydration and freezing stress (Danyluk et al. 1998). Most studies on dehydrins were performed in the laboratory and only a few field experiments have been done.

The objective of this work was to detect heat-stable COR proteins characteristic of hardened winter wheat plants at different phases of their ontogeny, to check if they related to dehydrins and to study the accumulation and disappearance of these proteins as related to the development and loss of plant cryotolerance under field conditions.

Materials and methods. Plants of 3- to 12-day-old etiolated seedlings and crowns of the winter wheat cultivar D 1(2) were used. This genotype is winter-resistant and highly productive under the climatic conditions of the forest steppes of eastern Siberia (Peshkova and Dorofeev 1998).

Seedlings were grown on moist filter paper in a thermostat at 20 C for 3 days and then cold acclimated at 4 C in darkness for 9 days. Shoots were cut at the end of days 1, 3, 7, and 9 and used for protein analysis. Unhardened 3-day-old seedlings were used as a reference.

For field experiments, seeds were sown at 5, 15, and 25 August and 5 September. Crowns or etiolated underground stem segments (depending on the phase of plant development) were sampled every 15 days throughout the entire period of spring growth and also in the middle of October. All treatments were performed in triplicate. Plants sown in August had time to tiller before winter, whereas plant sown on 5 September developed only to the three-leaf stage. Plants sown on 5 August developed more than five tillers, those on 15 August had three to five tillers, and those on 25 August had one or two tillers. Autumn growth of plants sown at different times lasted for 77, 68, 58, and 47 days, respectively.

In the Irkutsk region, the coldest month is January with an average daily temperature varying from -21 to -28 C. In the year of our field experiments, a stable snow cover was established on 13 November at a depth of 19 cm. After snow thaw at the end of March/beginning of April, the daily air temperature varied between 12 C to -17 C (Fig. 22). During this period, the temperature of the soil surface could change as much as 40°C during a single day (from a night temperature of -13 C to a day temperature of 27 C).

Winter hardiness was determined as the number of plants surviving in spring. Three sites for plant counting, with a total area of 1 m2, were marked in autumn on each experimental plot.

Water-soluble proteins were extracted from seedlings and crowns. Plant material (3.5 g) frozen in liquid nitrogen was ground into a powder with a mortar and pestle. Proteins were extracted with 100 mm Tris-HCl, ph 7.6, containing 10 mm dithiothreitol, 10 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride (Sigma, USA). The homogenate obtained was centrifuged at 17,000 x g for 15 min using the angle rotor of a K-24 centrifuge (Janetzki, Germany). To isolate heat-stable proteins, the supernatant was boiled for 20 min in a water bath and then centrifuged at 17,000 x g for 15 min to remove insoluble proteins (Lin et al. 1990). Total and heat-stable proteins were precipitated with 10 % trichloroacetic acid and separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978). On each lane, 15 µg of protein were loaded.

Proteins were subjected to SDS-PAGE using a mini-Protean page cell (Bio-Rad, USA) according to manufacturer's directions. Western blotting and immunodetection were as described by Timmons and Dunbar (1990) using anti-dehydrin primary antibody (1:1,000 dilution) kindly provided by T.J. Close (Close et al. 1993). In order to examine the specificity of antibodies, we used the antiserum preblocked with dehydrin consensus peptide (Werner-Fraczek and Close 1998). Density tracings were produced using a MD-100 densitometer (Carl Zeiss, Germany). All experiments were performed in three replicates. The figures illustrate representative electrophoregrams. Table 3 represents the average numbers of surviving plants for 6 years (1990-96) and their standard errors.

 

Results. Plants sown at different times differed in the survival (Table 3). Cold acclimation of 3-day-old winter wheat seedlings at 4 C resulted in the accumulation of several heat-stable proteins with molecular weights of 209, 196, 169, 66, 50, and 41 kDa. In unhardened seedlings, these proteins were either absent or present in minor amounts (Fig. 23). Some heat-stable proteins characteristic of hardened seedlings (with molecular weights of 209, 196, 66, and 50 kd) also were found in plants cold-acclimated under field conditions (Fig. 24A). With the approach of winter (30 October), the qualitative pattern of heat-stable proteins was similar in plants of different ages and did not change throughout the entire winter and spring until April (Fig. 24A and B). Before winter, the younger plants (sown on 5 September) accumulated less heat-stable proteins than plants sown on 25 or 15 August. Thus, morphologically, more-advanced plants (sown on 15 August) contained more heat-stable proteins (Fig. 24A). At the beginning of April (after snow thaw), quantitative changes in HMW heat-stable proteins occurred. By the middle of April (sampling on 16 April, 1996), differences between plants sown at different times became more distinct. By this time, the pattern of heat-stable proteins was the opposite of that observed in autumn. Thus, plants sown earlier, which accumulated more heat-stable proteins before the onset of winter, lost these proteins in spring more rapidly and returned to the unhardened state. In contrast, the content of heat-stable proteins in younger plants was higher than in morphologically more advanced plants (Fig. 25B). In May, no qualitative or quantitative differences were observed between plants sown at different times, except for a 50-kd protein. However, the differences in the amounts of this protein were also small (Fig. 25C).

Sampling on 16 April and 4 May was done after a considerable increase in the average daily temperature (see Fig. 22) and thaw. To elucidate whether the decrease in the content of heat-stable proteins was related to increased temperature, we sampled crowns on 2 April, 1996, when the soil was not yet thawed. Some samples were immediately frozen in liquid nitrogen, whereas other samples were kept for a day at room temperature.

We found no difference between heat-stable proteins between the plants sown at different times when samples were analyzed immediately after sampling (Fig. 25A), but this difference appeared after the sample was warmed for a day (Fig. 25B). Late-sown plants (25 August and 5 September) contained more heat-stable proteins with molecular weights of 209, 196, 66, and 50 kDa, but plants sown earlier (5 and 15 August) lost the heat-stable proteins characteristic of hardened plants more rapidly. When plants were sown later, they had time to produce only one or two tillers or they were at the three-leaf phase. Such plants survived much better than plants sown on 5 and 15 August (see Table 3).

Western-blotting revealed that heat stable proteins with molecular weights of 209, 196, 66, and 50 kDa both in the seedlings and crowns were related to dehydrins (Fig. 26). None of these proteins were detected on immunoblots developed with the serum preblocked with a dehydrin consensus peptide (data not shown).

We also found another protein related to dehydrin with a content higher in both hardened seedlings and crowns with the molecular weight of 41 kDa (Figs. 26A and B). We could not find it within the proteins from crowns stained by Coomassie (Fig. 26A), probably due to it small content (Fig. 26B).

Discussion. Cold acclimation is a complex biochemical process controlled by numerous cold-regulated genes. These genes have been identified in diverse plant species (Guy 1990, Alberdi and Corcuera 1991, Hughes and Dunn 1996, Thomashow 1998). We were interested in elucidating whether or not plant age at the onset of winter affected plant survival and whether proteins characteristic of plant hardiness were present in spring. We performed both laboratory and field experiments. In the field, the plant is affected not only by low temperature but also by a whole complex of environmental factors. Therefore, comparing model laboratory conditions with those that exist in nature is essential.

We detected some heat-stable proteins characteristic of hardened winter wheat plants with molecular weights of 209, 196, 66, and 50 kd. These polypeptides were present in both wheat seedlings hardened in the laboratory and in tillering plants or plants at the three-leaf stage acclimated under field conditions (Fig. 23 and Fig. 25). The synthesis and accumulation of these proteins evidently did not depend on the plant developmental phase, which confirms the data of other researchers (Perras and Sarhan 1989, Houde et al. 1992). The mechanism triggering the synthesis of cold regulated proteins did not depend on plant irradiation and was determined by drop in temperature (Uemura et al. 1996).

Heat-stable polypeptides of hardened winter wheat are very hydrophilic, stable during boiling, and their synthesis is activated by low temperature. Similar properties are characteristic of WCS proteins synthesized by hardened wheat plants (Houde et al. 1995). The WCS120 family is characteristic of frost-resistant cereals and includes five polypeptides with molecular weights of 40, 50, 66, 180, and 200 kDa. The contents of the 50- and 66-kDa proteins are considerably higher than those of other WCS proteins, which agrees with our results for heat-stable proteins (Fig. 23 and Fig. 25) and dehydrins (Fig. 26). However, the molecular weights of the other three polypeptides of this family differ from those of the proteins in our plant material. These differences can be attributed to the low accuracy of estimating molecular weights in electrophoretic studies or by protein polymorphism within the species. Of note is the fact that the apparent molecular mass of cold stress proteins may differ strictly from the mass predicted by its sequence (Houde et al. 1992, Danyluk et al. 1998). So far, we cannot definitely conclude that all the COR proteins we described belong to the WCS120 family. Some authors have noted a similarity between these proteins and dehydrins (Ouellet et al. 1993, Sarhan et al. 1997). We also showed that heat-stable proteins accumulated in both seedlings in the laboratory and crowns in the field supposed are dehydrins.

The accumulation of heat-stable polypeptides observed during the hardening of winter wheat seedlings is in agreement with the results obtained by other researchers (Lin et al. 1990, Alberdi and Corcuera 1991, Thomashow 1998). A similar response of plant cells to low-temperature stress was observed earlier in the different plants (Neven et al. 1993, Wolfraim et al. 1993, Gilmour et al. 1996, Uemura et al. 1996, Ismail et al. 1999).

Before winter, the pattern of the COR proteins in crowns was similar in plants of all ages, whereas the amounts of these proteins was higher in more developed plants (sown on15 August) than in younger plants (sown on 5 September). When soil and air temperature increases in the spring, overwintering cereals break dormancy and resume their growth and development. Plants protein patterns are changed under conditions favorable for growth. An increase in temperature results in the loss of the COR proteins specific to hardened plants. This conclusion is in agreement with the results of other researchers (Guy 1990, Houde 1992). Plants of different ages (different times of sowing) lost COR proteins at different rates (Fig. 25B and C). As far as we know, there is no such information in the literature. Plants sown earlier (5 and 15 August) lost COR proteins more rapidly and tolerated winter poorly. On the other hand, in younger, easily overwintering plants (sown on 25 August or 5 September), the content of heat-stable COR proteins was reduced more slowly. In all plants, HMW COR proteins were the first to disappear (Fig. 25).

Frost resistance in plants is determined by numerous factors among which are the content of soluble sugars and other cryoprotectants, desaturation of fatty acids, ultrastructural and biochemical changes in biomembranes. We speculate that COR proteins with molecular weights of 209, 196, 66, and 50 kDa, characteristic of hardened winter wheat plants, play an important role in protecting the plant against winter thaws and in early spring. These periods determine survival of cereal plants during the winter when dramatic temperature changes result in plant death.

The COR proteins we found in hardened winter wheat plants evidently can either protect plants against dehydration or are cryoprotectants. It is conceivable that they can serve as antinucleators, preventing intra-cellular ice formation. Further characterization of these heat-stable COR polypeptides will aid in the understanding of their role in the development of plant tolerance to low temperature.

Thus, both the seedlings and crowns accumulate the heat-stable proteins, presumably dehydrins, under the hardening to low temperature. These proteins were found at high levels before and during the winter in the crowns of winter wheat sown on different days. In the spring, dramatic changes in the amount of these proteins were observed. Younger plants, which survived the winter better, lost it more slowly. Morphologically more advanced plants, which tolerated winter badly, returned to a protein pattern characteristic of unhardened plants more rapidly, and that may be the reason of rapid decline of such plants.

Acknowledgments. This research was funded by a grant from the Russian Foundation of Basic Research (project 99-04-48121). We sincerely thank Dr. T.J. Close for his generous gift of the dehydrin antibody.

References.

• Alberdi M and Corcuera LJ. 1991. Cold acclimation in plants. Phytochem 30:3177-3184.
• Bohnert HJ, Nelson DE, and Jensen RG. 1995. Adaptations to environmental stresses. Plant Cell 7:1099-1111.
• Campbell SA and Close TJ. 1997. Dehydrins: genes, proteins, and associations with phenotypic traits. New Phytol 137:61-74.
• Close TJ and Lammers PJ. 1993. An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins. Plant Physiol 101:773-779.
• Close TJ, Fenton RD, and Moonan F. 1993. A view of plant dehydrins using antibodies specific to the carboxy terminal peptide. Plant Mol Biol 23:279-286.
• 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 commonality 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.
• Dure L. 1993. Structural motifs in lea proteins. In: Current topics in plant physiology: plant responses to cellular dehydration during environmental stress (Close TJ and Bray EA eds). ASPP, Rockville, MD. Pp. 91-103.
• Esen AA. 1978. A simple method for quantitative, semi-quantitative and qualitative assay of protein. Anal Biochem 89:264-273.
• Galinski EA. 1993. Compatible solutes of halophilic eubacteria: molecular principles, water-solute interactions, stress protection. Experientia 49:487-495.
• Gilmour SJ, Lin CT, and Thomashow MF. 1996. Purification and properties of Arabidopsis thaliana COR (cold-regulated) gene polypeptides COR15am and COR6.6 expressed in Escherichia coli. Plant Physiol 111:293-299.
• Guy CL. 1990. Cold acclimation and freezing tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41:187-223.
• Hare PD, Cress WA, and van Staden J. 1998. Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21:535-553.
• 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.
• Houde M, Dhindsa RS, and Sarhan F. 1992. A molecular marker to select for freezing tolerance in Gramineae. Mol Gen Genet 234:43-48.
• Hughes MA and Dunn MA. 1996. The molecular biology of plant acclimation to low temperatures. J Exp Bot 47: 291-305.
• 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.
• Labhilili M, Joudrier P, and Gautier M-F. 1995. Characterization of cDNAs encoding Triticum durum dehydrins and their expression patterns in cultivars that differ in drought tolerance. Plant Sci 112:219-230.
• Lin C, Guo WW, Everson E, and Thomashow MR. 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol 94:1078-1083.
• Mantyla E, Lang V, and Palva ET. 1995. Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LTI and RAB18 proteins in Arabidopsis thaliana. Plant Physiol l07:141-148.
• Moons A, Bauw G, Prinsen E, van Montagu M, and Straeten DVD. 1995. Molecular and physiological responses to abscisic acid and salts in roots of salt sensitive and salt tolerant indica rice varieties. Plant Physiol 107:177-186.
• Mtwisha L, Brandt W, McCready L, and Lindsey GG. 1998. HSP12 is a LEA-like protein in Saccharomyces cerevisiae. Plant Mol Biol 37:513-521.
• Neven LG, Haskell DW, Hofig A, Li Q-B, and Guy CL. 1993. Characterization of a spinach gene responsive to low temperature and water stress. Plant Mol Biol 21:291-305.
• Ouellet F, Houde M, and Sarhan F. 1993. Purification, characterization and cDNA cloning of the 200 kDa protein induced by cold acclimation in wheat. Plant Cell Physiol 34:59-65.
• Pelah D, Wang W, Altman A, Shoseyov O, and Bartels D. 1997. Differential accumulation of water stress-related proteins, sucrose synthase, and soluble sugars in Populus species that differ in their water stress response. Physiol Plant 99:153-159.
• Perras M and Sarhan F. 1989. Synthesis of freezing tolerance proteins in leaves, crown, and roots during cold acclimation of wheat. Plant Physiol 89:577-585.
• Peshkova AA and Dorofeev NV. 1998. Winter hardiness formation of wheat depending on vegetation condition and mineral nutrition. Agric Chem 2:24-29.
• Robertson AJ, Weninger A, Wilen RW, Ping FU, and Gusta LV. 1994. Comparison of dehydrin gene expression and freezing tolerance in Bromis inermis and Secale cereale grown in controlled environments, hydroponics, and the field. Plant Physiol 106:1213-1216.
• 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.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.
• Thomashow MF. 1998. Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118:1-8.
• Weretilnyk E, Orr W, While TC, Iu B, and Singh J. 1993. Characterization of three related low temperature-regulated cdnas from winter Brassica napus. Plant Physiol 101:171-177.
• Werner-Fraczek J and Close TJ. 1998. Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map position. Theor Appl Genet 97:220-226.
• Wolfraim LA, Langris R, Tyson IL, and Dhindsa RS. 1993. cDNA sequence, expression, and transcript stability of a sold-acclimation specific gene, cas18, of alfalfa (Medicago falcata) cells. Plant Physiol 101:1275-1282.
• Yamaguchi-Shinozaki K and Shinozaki K. 1994. Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 236:331-340.

 

Changes in the content of dehydrins and RAB-proteins in winter wheat during autumn hardening, wintering, and spring dehardening. [p. 186-187]

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

Autumn hardening of winter plants results in the increase of plant cryotolerance and contributes to their winter survival (Guy 1990). Hardening is accompanied by a gradual dehydration and hormonal balance change towards ABA accumulation and ultrastructural cell reconstruction. These processes are largely regulated by the expression of the genes encoding stress proteins or COR polypeptides. Many of them belong to the LEA (late embryogenesis abundant) superfamily, dehydrins, and RAB polypeptides and are of particular interest because of their cryoprotective properties (Leung and Giraudat 1998). The dehydrins and part of RAB proteins are thought to increase the ability of a cell to resist to extremes in pH and osmotic stress, contribute to solubilization, and protect other macromolecules and cell structures during the dehydration period (Close 1996, Ingram and Bartels 1996).

The study of the accumulation and disappearance of dehydrins during a life cycle was made on woody plants by Arora et al. (1997). The pattern of accumulation of dehydrins during the year coincided with seasonal fluctuations of cold resistance. However, as far as we know, the features of seasonal changes in the protein spectrum and content of dehydrins and in particular of RAB-polypeptides in wintering herbaceous plants under field conditions are not studied.

Our interest is to identify the proteins of these families in winter wheat plants of T. aestivum species grown in natural field conditions and study seasonal quantitative and qualitative changes in the content of these polypeptides.

Materials and methods. Crowns of the winter wheat cultivar Irkutskaia ozimaia were used in the study. All experiments were done in the field. Seed was sown on 25 August. Crowns were sampled in the field every 15 days throughout the entire period of autumn and spring growth (starting in November) and also monthly in the winter.

In our region of the Russian Federation, winter comes in the middle of October, when the mean diurnal temperature of the air is below 0°C. Spring starts in the middle of April, and the vegetation growth of plants resumes by the beginning of May.

Water-soluble, heat-stable, and total proteins were extracted from crowns as previously described by Lin et al (1990) with modifications (Stupnikova et al. 1998). Total and heat-stable proteins were separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978).

Proteins were separated by SDS­PAGE using a mini-Protean PAGE cell (Bio-Rad Laboratories, USA) according to manufacturer's instructions. Western blotting and immunodetection were as described by Timmons and Dunbar (1990) using anti-dehydrin and anti-RAB primary antibodies (1:1000 dilution), kindly provided by T. J. Close (University of California, Riverside, USA) and J. Mundy (Denmark, University of Copenhagen), respectively. Western blot images were analyzed by Sigma Scan Pro Software (Sigma Chemical, USA). The relative protein contents were estimated in arbitrary units. All the experiments were performed in three replicates. The figures illustrate representative immunochemical membranes.

Results and discussion. During the period of autumn hardening, all the plants accumulated dehydrins with molecular weights of 209, 196, 66, 50, and 41 kDa, which are related to RAB polypeptides. Synthesis of these proteins appears to be induced by dehydration and ABA. Moreover, they apparently have protecting functions characteristic for the families of dehydrins and RABs. The accumulation of the dehydrin/RABs was accompanied by an increase of frost-resistance (Fig. 27). The high content of these dehydrin/RABs during autumn and winter was stable until the beginning of April.

At the onset of winter, low-molecular dehydrins and RABs with molecular weights of 24, 22, 17, 15, and 12 kDa also accumulated (Fig. 27 and Fig. 28). The accumulation of low-molecular dehydrins and RABs is accompanied by a gradual decrease in frost hardiness (Guy 1990). The tendency for significant accumulation of these dehydrins and RABs by the end of winter may be connected with high-molecular proteins breaking into smaller units down to low-molecular under the impact of negative temperatures and with the de novo synthesis of the latter. Low-molecular dehydrins and RABs apparently perform a protective function in the course of damage accumulation during decreases in plant cryotolerance. All dehydrins and RABs disappeared in the spring during the period of plant dehardening (Fig. 28).

The pattern of accumulation/loss of the medium- and high-molecular proteins before winter and in the winter­spring period correspond with seasonal fluctuations in the cryotolerance of plants surviving winter, whereas low-molecular polypeptides accumulated during decreases in frost hardiness. High- and medium-molecular dehydrins/RABs are likely to participate both in stress-reaction and specialized plant adaptation, whereas low-molecular dehydrins and RAB-proteins apparently act only as proteins adaptation.

References.

• Arora R, Rowland LJ, and Panta GR. 1997. Chill-responsive dehydrins in blueberry: Are they associated with cold hardiness or dormancy transitions? Physiol Plant 101:8-16.
• Close TJ. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97:795-803.
• Esen AA. 1978. A simple method for quantitative, semi-quantitative and qualitative assay of protein. Anal Biochem 89:264-273.
• Guy CL. 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41:187-223.
• Ingram J and Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Ann Rev Plant Physiol Plant Mol Biol 47:377-403.
• Leung J and Giraudat J. 1998. Abscisic acid signal transduction. Ann Rev Plant Physiol Plant Mol Biol 49:199-222.
• Stupnikova IV, Borovskii GB, and Voinikov VK. 1998. Accumulation of hat-stable proteins in winter wheat seedlings during hypothermia. Rus J Plant Physiol 45:744-748.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Method Enzymol 182:679-688.

 

The impact of cold on the content of dehydrins and RAB proteins in the seedlings of spring and winter wheat. [p. 188-189]

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

Hypothermia causes various metabolic adjustments, which counteract low-temperature damage and induce freezing tolerance that is accompanied by gradual cell dehydration. Dehydration appears to activate the synthesis of various cold-regulated proteins (COR proteins) and trigger the production of abscisic acid, which, in turn, induces ABA-inducible genes (Yamaguchi-Shinozaki and Shinozaki 1994). COR genes encode stress proteins that are integrated into protein families based on sequence homology and have protective, transport, and regulatory functions. Stress proteins include dehydrins, RAB polypeptides (responsive to abscisic Acid, that is ABA-inducible), antifreeze proteins, HSPs, and uncoupling proteins. Interestingly, most of these have an unusually hydrophilic nature and many remain soluble upon boiling in dilute aqueous buffer (Thomashow 1999). Because the dehydration and biosynthesis of ABA are seemingly the key moments in cold adaptation, and also because most of the COR polypeptides are heat-stable, considerable attention has been given to the families of dehydrins and RAB proteins.

We have previously revealed a number of polypeptides with molecular weights of 209, 196, 169, 66, 50, and 41 kDa characteristic of hardened, winter wheat species (Stupnikova et al. 1998). Because of the correlation between content, accumulation of heat-stable proteins, and increased cryotolerance of seedlings, we assume that COR proteins play an important role in mechanisms of cell protection from the effects of low-temperature damage. Taking into account the above and their high stability during boiling, we suggest these revealed proteins belong to families of dehydrins and RAB proteins. One of the major functions of these polypeptides is to prevent local dehydration and protein denaturation (Close 1996).

Materials and methods. Three-day-old etiolated seedlings of the spring wheat cultivars Rollo, Drott, Angara-86, and Tyumenskaia-80; the medium, winterhardy, winter wheat cultivar Bezostaia-1; and the highly winter-hardy cultivars Irkutskaia ozimaia and Zalarinka were grown at 22 C. Unstressed plants were maintained under growth conditions for 1 day. A mild cold treatment (acclimation) was done by subjecting the seedlings to a temperature of 4 C for 9 days. Control and cold-treated seedling were compared at similar growth stages.

Water-soluble, heat-stable, and total proteins were extracted from seedlings as previously described by Lin et al. (1990) with modifications (Stupnikova et al. 1998). Total and heat-stable proteins were separated electrophoretically. Protein concentrations of the samples were determined according to Esen (1978).

Proteins were subjected to SDS-PAGE using a mini-Protean PAGE cell (Bio-Rad Laboratories, USA) according to the manufacturer's instruction. Western blotting and immunodetection were as described by Timmons and Dunbar (1990) using anti-dehydrin and anti-RAB primary antibodies (1:1000 dilution), kindly provided by T. J. Close (University of California, Riverside, USA) and J. Mundy (Denmark, University of Copenhagen). Western blot images were analyzed by Sigma Scan Pro Software (Sigma Chemical, USA). The relative contents of proteins were estimated in the arbitrary units. All the experiments were performed in three replicates. The figures illustrate representative immunochemical membranes.

Results and discussion. All spring and winter wheat plants that were hardened during 9 days at 4 C accumulated heat stable proteins with molecular weights of 209, 196, 169, 66, 50, and 41 kDa. The study indicated that these proteins are homologous to the polypeptides of the dehydrin family (Fig. 29), whereas proteins with molecular weights of 209, 196, 66, 50, and 41 kDa have immunochemical affinity to RABs (Fig. 30). This fact allowed us to suggest that first, their synthesis is induced by dehydration and ABA, and second, they apparently function by stabilizing membrane structures and hydrophobic patches of proteins.

Dehydrins with molecular weights of 209, 66, and 50 kDa were registered in control seedlings (Fig. 29), which is unusual for dehydrins because they were detected earlier in stressed plants (Sarhan et al. 1997). The abundance or absence of dehydrins in control plants apparently depends on the age of the wheat plants. We used young, four-diurnal plants. According to the literature, the maximum mRNA content of the dehydrins and proteins was marked in dry seeds and gradually disappeared after germination. We appear to have detected dehydrin residues.

A density analysis revealed a strong connection between the frost hardiness wheat of different cultivars and the relative content of dehydrins and RABs (Fig. 31). The greatest abundance of all dehydrins and RABs was detected in frost-resistant plants of the winter wheat cultivars Zalarinka and Irkutskaia ozimaia, and less frost-resistant Bezostaya-1. Interestingly, the medium, winter-hardy Bezostaya-1 has high-molecular proteins of intermediate weights of 196 and 169 kDa between frost-susceptible spring wheat seedlings of Rollo, Drott, Angara-86, and Tyumenskaia-80 and the highly, winter-hardy wheats Zalarinka and Irkutskaia ozimaia (Figs. 31B and C).

Apparently, the differences in the levels of plant freezing tolerance within one species are partly explained by differences in content and accumulation of cryoprotective compounds, e.g., such as dehydrins and RABs. These proteins possibly can be considered as frost-resistant markers of wheat species.

References.

• Close TJ. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97:795-803.
• Esen AA. 1978. A simple method for quantitative, semi-quantitative and qualitative assay of protein. Ann Biochem 89:264-273.
• Lin C, Guo WW, Everson E, and Thomashow MR. 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol 94:1078-1083.
• 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. 1998. Accumulation of heat-stable proteins in winter wheat seedlings during hypothermia. Russian J Plant Physiol 45:744-748.
• 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.
  Yamaguchi-Shinozaki K and Shinozaki K. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251-264.

 

 

VAVILOV INSTITUTE OF GENERAL GENETICS, RUSSIAN ACADEMY OF SCIENCES
Gubkin str. 3, 117809 Moscow, Russian Federation.

ENGELHARDT INSTITUTE OF MOLECULAR BIOLOGY, RUSSIAN ACADEMY OF SCIENCES
Vavilov str. 32, 117984 Moscow, Russian Federation.

T.I. Odintsova, E.D. Badaeva (Engelhardt Institute), E.N. Bilinskaya, and V.I. Pukhalsky.

Chromosome analysis and glutenin characterization in a wheat introgressive line, 224/2-96. [p. 190-191]

The wheat species T. kiharae (A^t^A^t^GGD^sq^D^sq^) is highly resistant to such widespread diseases as yellow rust and powdery mildew and its involvement in crosses with T. aestivum to introduce resistance genes into the common wheat genome seems promising. However, other genes (e.g., those responsible for grain quality) may be incorporated into the genome of common wheat together with the resistance genes.

The aim of this work was to study the karyotype and glutenins (HMW and D subunits) of an introgressive line 224/2-96 (T. aestivum/T. kiharae) resistant to yellow rust and powdery mildew. HMW-glutenin subunits are associated with technological quality of flour; D subunits are reported to be involved in termination of the glutenin polymeric chain. Therefore, their presence is correlated with poor quality.

Materials and methods. The introgressive line 224/2-96 was obtained by crossing T. aestivum line 353 (a mutant of the Mironovskaya cultivar with winter habit) and T. kiharae (pollen irradiation, 5 kRad) with subsequent self-pollination. Seed of the F8­F10 generation were examined. The standard C-banding procedure was used for chromosome analysis (Badaeva et al. 1990a). Chromosomes were identified on the basis of their C-banding patterns and classified according to the genetic nomenclature of common wheat (Badaeva et al. 1990b, Gill et al. 1991) and T. timopheevii (Badaeva et al. 1991) chromosomes. Propanol-soluble polymeric glutenins (70PI fraction) were obtained according to the procedure of Fu and Sapirstein (Fu and Sapirstein 1996). HMW-glutenin subunits were precipitated following the method of Marchylo et al. (1989), and the propanol-soluble fraction (60PS) was further separated by RP-HPLC on an Aquapore RP300 column with a linear acetonitrile gradient (23-48 %). Omega-gliadins and D-glutenin subunits were identified by SDS-PAGE and N-terminal sequencing.

Results and discussion. The investigation has shown that line 224/2/96 is stable with respect to chromosome number (2n = 6x = 42) (Badaeva et al. 2000). No intergenomic substitutions were observed. However, structural modifications of two chromosome pairs were detected. Thus, a distinct heterochromatic segment appeared in the short arm of chromosome 1A. Because such a band was absent from the parental line 353 karyotype, we may assume that chromosome 1A was involved in translocation. The second rearrangement occurred between 6B and an unidentified chromosome of either common wheat or T. kiharae. Chromosome 6B in the karyotype of introgressive line 224/96 has an increased length of the long arm, which contains a small C-band in the middle of the euchromatic region. However, the absence of other markers obstructs identification of the second chromosome involved in translocation.

The comparison of electrophoretic patterns of HMW-glutenin subunits in line 224/2/96, T. kiharae, and line 353 showed that the pattern of the introgressive line was more similar to that of line 353. The same is true for omega-gliadins. However, D-glutenin subunits of line 224/2/96 were similar to those of T. kiharae. To further characterize the glutenin fraction, propanol-soluble glutenins enriched in HMW- and D-glutenin subunits were separated by HPLC, and the obtained fractions analyzed by SDS-PAGE and N-terminal sequencing. D1 and D2 glutenin subunits were identified on the basis of their N-terminal sequences and were as follows: KELQSPQQSF for D1 and AEQLNPSNKE for D2. Glutenin subunit D3 was likely to be present both in line 224/2/96 and the parental form T. kiharae. The comparison of HPLC-patterns of showed that, in line 224/2/96, the portion of D1 and D2 subunits relative to HMW was higher than in T. kiharae. The results obtained indicate that the technological properties of flour obtained from line 224/2/96 may be comparable with those of T. kiharae. However, this suggestion needs further confirmation by direct rheological measurements.

This work was supported in part by the State Program 'Frontiers in Genetics'.

References.

• Badaeva ED, Boguslavsky RL, Badaev NS, and Zelenin AV. 1990a. Intraspecific chromosomal polymorphism of Triticum araraticum (Poaceae) detected by C-banding technique. Plant Syst Evol 169:13-24.
• Badaeva ED, Prokofieva ZD, Bilinskaya EN, Obolenkova LA, Solomatin DA, Zelenin AV, and Pukhalskyi VA. 2000. Cytogenetic analysis of hybrids resistant to yellow rust and powdery mildew obtained by crossing common wheat (Triticum aestivum, AABBDD) with wheats of the Timopheevi group (AtAtGG). Russian J Genet 36:1401-1410.
• Badaeva ED, Sozinova LF, Badaev NS, Muravenko OV, and Zelenin AV. 1990b. "Chromosomal passport" of Triticum aestivum L. em Thell. cv. Chinese Spring and standardization of chromosomal analysis of cereals. Cereal Res Commun 18:273-281.
• Gill BS, Friebe B, and Endo TR. 1991. Standard karyotype and nomenclature system for description of chromosome bands and structural aberrations in wheat (Triticum aestivum). Genome 34:830-839.
• Badaeva ED, Budashkina EB, Badaev NS, Kalinina NP, and Shkutina FM. 1991. General features of chromosome substitutions in Triticum aestivum x T. timopheevii hybrids. Theor Appl Genet 82:227-232.
  Fu BX and Sapirstein HD. 1996. Procedure for isolating monomeric proteins and polymeric glutenin of wheat flour. Cereal Chem 73:143-152.
• Marchylo BA, Kruger JE, and Hatcher DW. 1989. Quantitative reversed-phase high-performance liquid chromatographic analysis of wheat storage proteins as a potential quality prediction tool. J Cereal Sci 9:113-130