ITEMS FROM THE RUSSIAN FEDERATION

SIBERIAN INSTITUTE OF PLANT PHYSIOLOGY AND BIOCHEMISTRY

Siberian Division of the Russian Academy of Sciences, Lermontov str., 132, Irkutsk-33, P.O Box 1243, Russian Federation, 664033.

The function of different mitochondrial respiratory-chain pathways in winter wheat mitochondria during short-term cold stress and hardening. [p. 108-110]

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

Plant mitochondria have a branched respiratory chain and, in addition to the main cytochrome pathway, have an alternative pathway that depends on the functioning of alternative cyanide-resistant oxidase (AOX) (Vanlerberghe and McIntosh 1997). Plant mitochondria also are able to oxidize exogenous NAD(P)H because of the presence of additional NAD(P)H dehydrogenases in their structure (Soole et al. 1990; Soole and Menz 1995; Moller and Rasmusson 1998). Recently, a number of proteins that effect mitochondrial activity were found and characterized. Among these are plant uncoupling mitochondrial proteins (plant UCPs) (Ricquier and Bouillaud 2000) and the stress protein CSP 310 (Voinikov et al. 1998), which cause uncoupling of oxidative phosphorylation in mitochondria. AOX (Takumi et al. 2002), WhUCP (Murayama and Handa 2000), and CSP 310 (Kolesnichenko et al. 2000) are present in the mitochondria of winter wheat. Some of these proteins, such as AOX and CSP 310, are induced by cold stress in winter wheat, but others (WhUCP) are not. Although WhUCP is not induced by cold stress in winter wheat, its homologues in other plant species were shown to be induced by cold stress (Laloi et al. 1997; Maia et al. 1998; Ito 1999; Nantes et al. 1999). The main functions of these proteins were established for animals and proposed for plants are thermogenesis, participation in defense against oxidative stress, and regulation of cell metabolism (Sluse and Jarmuszkiewicz 2002). On the other hand, mechanisms that control the different electron-transport pathways in mitochondrial respiration under different stress conditions have not been studied in detail. Using inhibitor analysis that blocks terminal oxidases or respiratory-chain complexes, we studied the role of individual mitochondrial respiratory chain pathways in total mitochondrial respiration to learn how the different electron-transport pathways function in cold-resistant, winter wheat mitochondria during short-term cold stress and hardening.

Materials and methods. Three-day-old etiolated shoots of the winter wheat cultivar Zalarinka were germinated on moist paper at 26 C. Shoots were cold-stressed at -1 C for 1 h or were hardened at 4 C for 7 days. Mitochondria were extracted from seedling shoots by differential centrifugation (Pobezhimova et al. 1996). Isolated mitochondria were resuspended in a medium of 40 mM MOPS-KOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2. Mitochondrial activity was recorded polarographically at 27 C using a closed platinum electrode in a 1.4-ml volume cell (Estabrook 1967). The reaction mixture contained 125 mM KCl, 18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. 10 mM malate in the presence of 10 mM glutamate, 8 mM succinate in the presence of 5 mM glutamate and 1 mM NADH were used as oxidation substrates. During succinate and NADH oxidation, 3 mkM rotenone was added to the incubation medium. During NADH oxidation, 0.06 mM CaCl2 was added to incubation medium. The concentrations of inhibitors of the respiratory chain were antimycin A (A-A) (20 mkM), BHAM (1 mM), KCN (0.4 mM), and CSP 310 antiserum (1 mg/mL). Polarograms were used to calculate the rates of phosphorylative respiration (state 3), nonphosphorylative respiration (state 4), respiration control by Chance-Williams (RC), and the ADP:O ratio (Estabrook 1967). The concentration of mitochondrial protein was analyzed according to Lowry et al. (1951). All experiments were performed on 3-6 separate mitochondrial preparations. The data obtained were analyzed statistically and arithmetic means and standard errors were determined.

Results and discussion. We studied the mitochondrial respiratory-chain function of winter wheat during short-term low temperature stress and hardening using different oxidation substrates. When using succinate and NADH as oxidation substrates, rotenone, which blocks electron transfer through complex I of the mitochondrial respiratory chain, was added the mitochondrial-incubation medium. When using malate as oxidation substrate, winter wheat mitochondria isolated from control seedling shoots were well coupled (Table 1). After short-term low-temperature stress, the rates of state-3 and state-4 respiration increased by 19.2 % and 43.8 %, respectively, and the respiratory-control coefficient (RC) decreased (17.3 %) when compared to the control (Table 1). This data shows that these mitochondria were uncoupled. On the other hand, mitochondria isolated from hardened winter wheat seedling shoots had a lower rate of state-3 and state-4 respiration than the control mitochondria and changes in their RC coefficient and ADP:O ratio were to a lesser degree (13 % for state 3 and 11 % for state 4) (Table 1). When succinate was used as an oxidation substrate, we found that neither short-term low-temperature stress nor cold hardening influenced the degree of coupling of isolated mitochondria (Table 1). When NADH was the oxidation substrate, results were similar to those of succinate; no significant difference between mitochondria isolated from control, stressed, and hardened shoots (Table 1). Thus, cold stress caused significant changes only in the activity of malate-oxidizing mitochondria but did not influence succinate- and NADH-oxidizing mitochondria. Short-term cold stress caused more pronounced changes in mitochondria energetic activity then cold hardening.

The participation of the main cytochrome and alternative pathways in mitochondrial respiration was studied by adding an oxidation substrate, mitochondria, and ADP to the polarographic cell. When mitochondria were in state-4 respiration, antimycin A, BHAM, and anti-CSP 310 antiserum or KCN were added to the polarographic cell. We found that malate-oxidizing mitochondria isolated from control, stressed, and hardened seedling shoots differed in their reaction to inhibitor addition. Antimycin A in addition to control mitochondria caused ~ 50 % decrease of oxygen consumption. In mitochondria isolated from stressed plants, this treatment caused only ~ 30 % decrease (Figure 1A). Cold shock caused ~ 20 % increase of antimycin A-resistant mitochondrial respiration. In mitochondria isolated from hardened plants, addition of antimycin A caused ~ 65 % decrease in oxygen consumption. Consequent addition of BHAM to mitochondria isolated from control and hardened plants inhibited oxygen consumption up to 25 % from state-4 respiration but in mitochondria isolated from stressed plants, this treatment inhibited oxygen consumption only up to 33 % (Figure 1A). Therefore, we can conclude that in control mitochondria about 25 % of the respiration is antimycin A- and BHAM-resistant and that this part of mitochondria respiration increased during short-term low-temperature stress but was at the level of the control plants during cold hardening. The residual mitochondrial oxygen consumption was fully inhibited by consequent addition of anti-CSP 310 antiserum or KCN, so we can conclude that this residual respiration is involved with CSP 310 function.

Adding antimycin A to succinate-oxidizing mitochondria caused ~ 90 % inhibition of oxygen consumption (Fig. 1B). The consequent addition of BHAM to control mitochondria fully inhibited oxygen consumption. Despite the absence of cold-shock influence on total mitochondrial activity (Table 1), this treatment caused an increase of antimycin A-resistant respiration to ~ 20 % of that of state-4 respiration. Consequent addition of BHAM nearly inhibited mitochondrial respiration (Figure 1B). Cold hardening caused an increase of antimycin A-resistant respiration to ~40 % that of state-4 respiration. This antimycin A-resistant respiration also was nearly inhibited by BHAM addition (Figure 1B). We conclude that in succinate-oxidizing winter wheat mitochondria only two electron-transport pathways function, the main cytochrome pathway and an alternative antimycin A-resistant oxidase. Both cold shock and especially cold hardening caused an increase in this alternative pathway.

In NADH-oxidizing control winter wheat mitochondria, the addition of antimycin A caused ~ 80 % decrease of oxygen consumption (Figure 1C). Consequent BHAM addition fully inhibited oxygen consumption in control mitochondria, but this addition and even the consequent addition of anti-CSP 310 antiserum did not fully inhibit oxygen consumption in mitochondria isolated from stressed plants. The residual respiration in this case was about 10 %. Based on these data, we concluded that in succinate- and NADH-oxidizing mitochondria the main part of respiration depends on the functioning of the main cytochrome respiratory chain pathway (77 % and 91 %, accordingly) but only ~ 50 % of respiration depends on this pathway function in malate-oxidizing mitochondria.

Wheat mitochondria have different electron transport pathways. One is an alternative KCN- and antimycin A-resistant oxidase. In addition to this pathway, different types of uncoupling proteins recently were found in plant mitochondria. The plant stress protein CSP 310 is one (Voinikov et al. 1998). Data obtained from inhibitor analyses agree with that about the influence of exogenous CSP 310 on different mitochondrial respiratory-chain complex function (Grabelnych et al. 2001). The effect of CSP 310 addition to isolated plant mitochondria was detected at complex I function but was not detected in the functioning of other respiratory chain complexes. Now, we can show that the main contribution to mitochondrial respiration of the CSP 310-pathway that was inhibited by anti-CSP 310 addition was detected during malate oxidation (25 %).

Because antimycin A addition blocks electron transfer through Q-cycle, i.e., inhibits the main cytochrome respiratory chain pathway, we can conclude that residual mitochondrial respiration depends on the functioning of alternative pathways. Therefore, during malate oxidation, the main cytochrome pathway contributes ~ 50 % to the total mitochondria respiration. The residual 50 % depends on alternative oxidase (25 %) and CSP 310 (25 %) functioning (Table 2). Cold shock caused about a two-fold decrease in the main cytochrome pathway and increased the contribution of alternative pathways. On the other hand, cold hardening caused an increase in the cytochrome pathway contribution and decreased the contribution of alternative pathways in mitochondrial respiration (Table 2).

During succinate oxidation, the main part of mitochondrial respiration depends on the main cytochrome pathway function (about 90 %). At the same time, during succinate oxidation, short-term low-temperature stress and especially cold hardening caused a significant increase of alternative oxidase function. In NADH-oxidizing winter wheat mitochondria isolated from control plants, the main part of mitochondrial respiration also depends on cytochrome pathway function (about 77 %). Both cold shock and hardening did not significantly influence the contribution of different pathways in NADH-oxidizing mitochondria. Concurrently, we also detected an increase of residual mitochondrial respiration after antimycin A and anti-CSP 310 addition up to 10 % in these conditions (Table 2). In our opinion, this fact could depend on the function of external rotenone-insensitive and antimycin A-insensitive NADH-cytochrome c reductase (Soole et al. 1990).

Based on our data, we conclude that the contribution of the different mitochondrial electron-transport pathways significantly depends on the oxidized substrate. Short-term cold stress and cold hardening differ in their influence on the different electron transport pathways in winter wheat mitochondria.

Acknowledgments. The work was possible, in part, with the support of the Russian Foundation of Basic Research (projects 00-04-48093 and 02-04-06096) and the Siberian Division of Russian Academy of Sciences Youth Grant (project 78).

References.

• Estabrook RW. 1967. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratio. Meth Enzymol 10:41-47.
• Grabelnych OI, Pobezhimova TP, Kolesnichenko AV, and Voinikov VK. 2001. Complex I of winter wheat mitochondria respiratory chain is the most sensitive to uncoupling action of plant stress-related uncoupling protein CSP 310. J Therm Biol 26:47-53.
• Ito K. 1999. Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus). Plant Sci 149:167-173.
• Kolesnichenko AV, Zykova VV, Grabelnych OI, Sumina ON, Pobezhimova TP, and Voinikov VK. 2000. 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 Therm Biol 25:245-249.
• Laloi M, Klein M, Riesmeier JW, Muller-Rober B, Fleury Ch, Bouillaud F, and Ricquier D. 1997. A plant cold-induced uncoupling protein. Nature 389:135-136.
• Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with folin phenol reagent. J Biol Chem 193:265-275.
• Maia IG, Benedetti CE, Leite A, Turcinelli SR, Vercesi AE, and Arruda A. 1998. AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein. FEBS Lett 429:403-406.
• Moller IM and Rasmusson AG. 1998. The role of NADH in the mitochondrial matrix. Trends Plant Sci 3:21-27.
• Murayama S and Handa H. 2000. Isolation and characterization of cDNAs encoding mitochondrial uncoupling proteins in wheat: wheat UCP genes are not regulated by low temperature. Mol Gen Genet 264:112-118.
• Nantes IL, Fagian MM, Catisti R, Arruda P, Maia IG, and Vercesi AE. 1999. Low temperature and aging-promoted expression of PUMP in potato tuber mitochondria. FEBS Lett 457:103-106.
• 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.
• Ricquier D and Bouillaud F. 2000. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem J 345:161-179.
• Sluse FE and Jarmuszkiewicz W. 2002. Uncoupling proteins outside the animal and plant kingdoms: functional and evolutionary aspects. FEBS Lett 510:117-120.
• Soole KL, Dry IB, and Wiskich JT. 1990. Oxidation of NADH by plant mitochondria: kinetics and effects of calcium ions. Physiol Plantarum 78:205-210.
• Soole KL and Menz RI. 1995. Functional molecular aspects of the NADH dehydrogenases of plant mitochondria. J Bioenerg Biomembr 27:397-406.
• Takumi S, Tomioka M, Eto K, Naydenov N, and Nakamura C. 2002. Characterization of two non-homoeologous nuclear genes encoding mitochondrial alternative oxidase in common wheat. Genes Genet Syst 77:81-88.
• Vanlerberghe GC and McIntosh L. 1997. Alternative oxidase: from gene to function. Ann Rev Plant Physiol Plant Mol Biol 48:703-734.
• 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 use of linoleic acid as an oxidation substrate by winter wheat mitochondria. [p. 112-114]

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

Free fatty acids (FFA) are effective uncouplers of oxidative phosphorylation depending on their protonophoric activity, which causes a significant increase in the conductance of the inner mitochondrial membrane. Some data shows that saturated FFA has less influence on mitochondrial membrane potential then unsaturated FFA (Penzo et al. 2002). In addition, saturated FFA can regulate mitochondrial uncoupling protein activity (Jezek et al. 1997; Jarmuszkiewicz et al. 1998; Costa et al. 1999; Hourton-Cabassa et al. 2002) and even expression of these proteins (Muzzin et al. 1999; Sbrassia et al. 2002).

The major FFA catabolic pathway in the cell is b-oxidation, which results in acetyl-CoA that can be completely oxidized by cell to CO2 and H2O via the Kreb's Acid Cycle. Intermediates of this cycle are the main mitochondrial respiration substrate (Schulz 1991). The FFA b-oxidation activity of this pathway significantly increases upon seed germination but dramatically decreases after photosynthesis establishment and during vegetative growth (Masterson and Wood 2000). FFA was used as an oxidation substrate during the very early stages of sunflower and lettuce seed germination (Salon et al. 1988; Raymond et al. 1992) and in potato storage organs (Theologis and Laties 1980). At the same time, data on the capability of wheat-seedling mitochondria to use FFA as an oxidation substrate and about the participation of different mitochondrial electron transport pathways in this process are lacking.

Thus, the aim of this study the function of winter wheat mitochondria during oxidizing of FFA and the participation of different mitochondrial electron-transport pathways in this process.

Materials and methods. Three-day-old, etiolated shoots of winter wheat cultivar Zalarinka were germinated on moist paper at 26 C. Mitochondria were extracted from seedlings shoots by differential centrifugation (Pobezhimova et al. 1996). The isolated mitochondria were resuspended in the following medium: 40 mM MOPS-KOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2. Mitochondrial activity was recorded polarographically at 27 C using a 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. Malate (10 mM) in the presence of glutamate (10 mM) and linoleic acid (0.056-750 mkM) were used as oxidation substrates. The concentrations of inhibitors of respiratory chain were rotenone (3 mkM), antimycin A (A-A) (20 mkM), BHAM (1 mM), and CSP 310 antiserum (1 mg/ml). Polarograms were used to calculate the rates of phosphorylative respiration (state 3), nonphosphorylative respiration (state 4), respiration control by Chance-Williams (RC), and the ADP:O ratio (Estabrook 1967). The concentration of mitochondrial protein was analyzed by Lowry method (Lowry et al. 1951). All the experiments were performed on three separate mitochondrial preparations. The data obtained were analyzed statistically and arithmetic means and standard errors determined.

Results and discussion. The amount of total FFA in winter wheat mitochondria is about 15 ng/mg of mitochondrial protein (0.056 mkM) and increases to ~40 ng/mg (0.15 mkM) after short-term cold shock (Vojnikov et al. 1983). In our experiments, we used physiological concentrations of FFA and higher concentrations (1-750 mkM).

In the first set of experiments, linoleic acid (LA) was added to malate oxidizing mitochondria in state 4 (Figure 2, 1). We found that LA did not influence mitochondrial oxygen uptake in the range of 0.056-5 mkM. At 10 mkM, LA increased oxygen uptake by 25 %. At 20 mkM, a 87 % increase of oxygen uptake was detected. Further increases in the LA concentration in the mitochondria incubation medium (20-60 mkM) did not cause further increases in state-4 respiration. On the other hand, adding 100 mkM or more LA caused at least a three-fold increase in mitochondrial oxygen uptake with a maximum at 500 mkM. The addition of 100 mkM LA caused an increase in the level of state-4 respiration up to that of state-3 respiration.

Similar results were obtained when the oxidizing of LA was the only oxidation substrate for mitochondria (Figure 2, 2). Physiological FFA concentrations and concentrations up to 5 mkM did not cause an increase in oxygen uptake by winter wheat mitochondria. At the same time, at a concentration of 10 mkM, mitochondrial oxygen uptake up to 43 % was detected. Higher LA concentrations caused increases in oxygen uptake by mitochondria. The maximum oxygen uptake by winter wheat mitochondria was at LA concentration of 500 mkM. The rate of uncoupled respiration (Figure 2, 1) and the rate of linoleic acid-supported respiration (Figure 2, 2) were equal; 50 mkM LA.

Our data show that wheat mitochondria can successfully use linoleic acid as respiration substrate. Therefore, we were interested in determining what mitochondrial electron-transport pathways participate in this process. By looking at the influence of different electron-transport pathway inhibitors on oxygen uptake during 100 mkM LA oxidation, we found that different mitochondrial electron-transport pathways participate in this process. The data indicate that ~31 % of oxygen consumption was inhibited by the addition of antimycin A, ~34 % was inhibited by BHAM addition, ~33 % was inhibited by rotenone addition, and 30 % was inhibited by anti-CSP 310 addition.

During the oxidizing of LA, our data show that electrons can transfer oxygen through all branches of the electron-transport chain. Because rotenone is a complex-I inhibitor, the part of mitochondrial respiration that is not inhibited by its addition could deal with the functioning of complex II and different rotenone-insensitive, internal NADH dehydrogenases (Moller 1997).

Antimycin A addition blocks electron transport through complex III and, after this treatment, only alternative CN-resistant oxidase (Vanlerberghe and McIntosh 1997) and CSP 310 (Kolesnichenko et al. 2002) still function. These results agree with data on the influence of BHAM, which is an inhibitor of alternative CN-resistant oxidase and anti-CSP 310 antiserum, and its addition inhibits oxygen consumption dependent on CSP 310 function. Therefore, the LA-dependent increase in oxygen consumption is involved with the functioning of all branches of mitochondrial electron transport chain, both phosphorylative and nonphosphorylative.

Hermesh et al. (1998) used very high concentrations (0.5­2 mM) of FFA when studing mitochondrial energetic activity and proposed that FFA effects depend on the FFA-dependent uncoupling of oxidative phosphorylation. We have shown that LA concentrations higher than 50 mkM mitochondria change their metabolism to oxidizing LA as an oxidation substrate, because the rate of LA-supported respiration becomes equal to the uncoupled rate after the addition of LA respiration during malate oxidation. The function of the main cytochrome pathway in such conditions could depend on oxidative phosphorylation uncoupling because FFA uncoupling activity causes an increase of oxygen consumption. In addition to this pathway, other alternative electron-transport pathways function during LA oxidation. Based on our data, winter wheat mitochondria can use LA as an oxidation substrate. Linoleic acid oxidation in these conditions depends on the functioning of all electron-transport pathways that exist in plant mitochondria.

Acknowledgment. This work was performed, in part, with the support of the Siberian Division of Russian Academy of Sciences Youth Grant (project 78).

References.

• Costa ADT, Nantes IL, Jezek P, Leite A, Arruda P, and Vercesi AE. 1999. Plant uncoupling mitochondrial protein activity in mitochondria isolated from tomatoes at different stages of ripening. J Bioenerg Biomembr 31:527-533.
• Estabrook RW. 1967. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratio. Methods Enzymol 10:41-47.
• Hermesh O, Kalderon B, and Bar-Tana J. 1998. Mitochondrial uncoupling by long chain fatty acyl analogue. J Biol Chem 1273:3937-3942.
• Hourton-Cabassa C, Mesneau A, Miroux B, Roussaux J, Ricquier D, Zachowski A, and Moreau F. 2002. Alteration of plant mitochondrial proton conductance by free fatty acids. Uncoupling protein involvement. J Biol Chem 277:41533-41538.
• Jarmuszkiewicz W, Almeida AM, Sluse-Goffart CM, Sluse FE, and Vercesi A. 1998. Linoleic acid-induced activity of plant uncoupling mitochondrial protein in purified tomato fruit mitochondria during resting, phosphorylating, and progressively uncoupled respiration. J Biol Chem 273:34882-34886.
• Jezek P, Costa ADT, and Vercesi AE. 1997. Reconstituted plant uncoupling mitochondrial protein allows for proton translocation via fatty acid cycling mechanism. J Biol Chem 272:24272-24278.
• Kolesnichenko AV, Pobezhimova TP, Grabelnych OI, and Voinikov VK. 2002. Stress-induced protein CSP 310: a third uncoupling system in plants. Planta 215:279-286.
• Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with Folin phenol reagent. J Biol Chem 193:265-275.
• Masterson C and Wood C. 2000. Mitochondrial b-oxidation of fatty acids in higher plants. Physiol Plantarum 109:217-224.
• Moller IM. 1997. The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria. Physiol Plantarum 100:85-90.
• Muzzin P, Boss O, and Giacobino JP. 1999. Uncoupling protein 3: its possible biological role and mode of regulation in rodents and humans. J Bioenerg Biomembr 31:467-473.
• Penzo D, Tagliapietra C, Colonna R, Petronilli V, and Bernardi P. 2002. Effects of fatty acids on mitochondria: implications for cell death. Biochim Biophys Acta 1555:160-165.
• Pobezhimova TP, 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.
• Raymond P, Spiteri A, Dieuaide M, Gerhardt and Pradet A. 1992. Peroxisomal b-oxidation of fatty acids and citrate formation by a particulate fraction from early germinating sunflower seeds. Plant Physiol Biochem 30:153-162.
• Salon C, Raymond P, and Pradet A. 1988. Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos. J Biol Chem 263:12278-12287.
• Sbrassia P, D'Adamo M, Leonetti F, Buongiorno A, Silecchia G, Basso MS, Tamburrano G, Lauro D, Federici M, Daniele ND, and Lauro R. 2002. Relationship between plasma free fatty acids and uncoupling protein-3 gene expression in skeletal muscle of obese subjects: in vitro evidence of a causal link. Clinical Endocrinol 57:199-207.
• Schulz H. 1991. Beta oxidation of fatty acids. Biochim Biophys Acta 1081:109-120.
• Theologis A and Laties GG. 1980. Membrane lipid breakdown in relation to the wound-induced and cyanide-resistant respiration in tissue slices. Plant Physiol 66:890-896.
• Vanlerberghe GC and McIntosh L. 1997. Alternative oxidase: from gene to function. Ann Rev Plant Physiol Plant Mol Biol 48:703-734.
• Vojnikov VK, Luzova GB, and Korzun AM. 1983. The composition of free fatty acids and mitochondrial activity in seedlings of winter cereals under cold shock. Planta 158:194-198.

Questioning the possible role of D-amino acids in wheat seedlings. [p. 115-117]

N.I. Rekoslavskaya, R.K. Salyaev, V.M. Sumzova, T.V. Kopytina, and A.M. Sobenin.

The D isomers of different amino acids (alanine, tryptophan, aspartate, glutamate, proline, and other amino acids) and their derivatives have been detected in plants (Bell 1980), but their possible physiological functions are unknown in plants. The presence of nonproteinogenic, D-amino acids in seeds and seedlings is believed to protect plant tissues from pathogens and parasites (Bell 1980).

D-amino acids are actively synthesized by bacteria and low fungi (Davies 1977). Alanine racemase is of great importance to bacteria because it supplies them with D-alanine from available L-alanine. Therefore, alanine racemase may be a key enzyme in the synthesis of the protective peptidoglucan layer of the cell wall. In some cases, the D-amino acids are abundant (Vicario et al. 1987).

Another mechanism by which D-amino acids are formed involves D-amino acid aminotransferase, which produces a diversity of D-amino acids. Perhaps the synergistic action of the two enzymes racemase and D-amino acid transferase accounts for the large amount of different D-amino acids that appear in bacterial cells and plant seedlings.

D-alanine and its dipeptide, D-alanyl-D-alanine, make up a considerable part of the nitrogen pool and probably play a significant part in regulation of nitrogen metabolism in bacteria. D-amino acids are not toxic in plants, perhaps because of neutralization via malonylation, acetylation, and glycosylation followed by compartmentalization in the vacuole. The bonding of D-amino acids with malonyl or acetyl moyeties may be hydrolyzed and reveal amino acids in intact form.

D-alanine and its derivatives in pea seedlings appeared during germination and disappeared on the 8th day of growth (Ogawa et al. 1973). D-alanyl-D-alanine and D-alanylglycine were found in rice seedlings and leaves, respectively (Manabe 1986; Manabe and Ohira 1983). Free and bound D-aspartic and D-glutamic acids were determined in pea seedlings (Ogawa et al. 1977). The N-malonyl-D-tryptophan content increased in leaves of tomato, potato, wheat, and other species during wilting and after drought during the period of recovery after osmotic stress (Rekoslavskaya et al. 1988).

All of these data would seem to indicate that synthesis of D-amino acids and their further conversion have ontogenetic, physiologic, and ecologic significance that is still unknown. As for N-malonyl-D-tryptophan, an acceptable hypothesis is that it functions as a precursor of the plant hormone indoleacetic acid, IAA (Rekoslavskaya et al. 2002). In reality, D-tryptophan has been demonstrated in a number of cases to be as active or even more active than L-tryptophan as an auxin substitute (Rekoslavskaya 1986).

Using D-tryptophan as an IAA precursor illustrates the idea that pools of amino acids for nonprotein synthesis can be created by means of the conversion of L-amino acids to D-amino acids. Direct competition for the amino acid between nonprotein syntheses and protein synthesis occurs in the process of growth and development.

Thus, the appearance of D-amino acids in plants apparently is nonrandom, uncontrolled, and physiological meaningless event, but the physiological significance of D-amino acids remains largely unclear and needs detailed study. We have investigated the content of amino acids in wheat seedlings in relation with some enzyme activities of amino acids metabolism different from protein biosynthesis have been done. The specific activity of racemase, transaminase, and UDPG-transferase were estimated in wheat seedlings during the study.

Materials and methods. The spring wheat cultivar Scala was used in this study. Procedures to determine racemase and transaminase activities were as described by Rekoslavskaya et al. (2002). UDPG-transferase activity was determined according to the modified method primarily described by Kowalczyk and Bandurski (1991). Briefly, 21 g of leaf, 44 g of stem, 5.6 g of young kernel, and 35.1 g of root tissue of green wheat shoots were harvested, ground with mortar and pestle in liquid nitrogen, and extracted with the buffer containing 0.25 M HEPES, 5 mM EDTA Na2, 0.1 % mercaptoethanol, and 0.025 % Triton X-100, pH 8.5. One mg of phenylmethylsulfonylfluoride was added to the ground material at the time of extraction in order to prevent protease activity. The homogenate was passed through four layers of cheesecloth and centrifuged at 10,000 x g for 20 min at 4 C. The activity of UDPG-transferase was estimated in the supernatant fraction of each sample. The reaction mixture contained as the substrate 5 mmol of indoleacetic acid (IAA), 5 mmol of UDPG as the cofactor, and in order to prevent the ribosomes activity, 10^-4^ M CaCl2 were added to 1 ml of supernatant. The reaction mixture was then incubated for 16 hours at 37oC. The reaction was stopped by adding of 1 ml of isopropanol. The activity of UDPG-transferase was determined as nmoles of substrate converted during 1 h/mg of protein. The IAA glucose ester content was determined after passing of reaction mixtures through a DEAE-cellulose (acetate form) minicolumn (10 x 20 mm) in 6 ml of eluates of 50 % isopropanol. The Ehrlich reagent was used in order to determine IAA-glucose content with calibration curve made with IAA. A D-amino acids kit was used (Sigma, USA). L-Amino acids were from Reachim (Russian Federation). The content of amino acids were determined on an amino-acid analyzer AAA-1 (Microtechna, Czech Republic).

Results and discussion. The amino-acid content of 7-day-old seedling are presented in Figure 3. The amino acids Glu, Ala, Val, Pro, Leu, and iLeu had the highest content of > 200 mg/g of fresh weight. The content of Asp was next highest, but the other amino acids were present at levels below 100 nmol/g of fresh weight. Free Try did not contribute any significant content of free amino acids, but the sum of free and bound malonyl D-Try content was nearest to the content of Glu or even greater in seedlings sustaining wilting; 890 nmol/g of fresh weight (Rekoslavskaya et al. 1988).

The appearance of D-amino acids, and especially D-Try, during germination and growth of etiolated seedlings in the dark was shown previously (Rekoslavskaya et al. 2002). The activity of tryptophan racemase was found in the cytosol and etioplast fractions of wheat seedlings. The enzyme was isolated and some biochemical characteristics were studied, but the substrate specificity was broader and racemase used other amino acids as substrates (Table 3).

As shown in Table 3, the chiralic purity of D- or L-amino acids used were estimated with D-amino acid oxidase from hog kidneys (Sigma, USA) or with L-amino acids oxidase from snake venom (Sigma, USA). When D- or L-amino acids were treated with the enzyme preparation from wheat seedlings prepared as described earlier (Rekoslavskaya et al. 2002), we observed higher enzyme activities than in the case of either D- or L-tryptophan. For example, the specificity to Ala, Thr, Val, or Ser was about 5.8 or 4.5 times higher than to Try. The activity of transaminase was higher if Ala, Ser, Val, and some other amino acids were exploited in the study in comparison to Try. Therefore, it might be concluded that there was racemase and transaminase with broad substrate activities in wheat seedlings with some preference to amino acids structurally related to Ala.

About half of the amino acids is in the form of D-enantiomers in etiolated wheat seedlings. The content of D- and L-amino acids in 7-day-old wheat seedlings were 233.4 ± 34.0 and 194.8 ± 9.2 nmol/100 seedlings, respectively. We found two pools of amino acids in growing wheat seedlings and question why half of the amino acids in wheat are in a nonproteinogenic form that is not available for the synthesis of protein.

We tried to explain the appearance of D-Try in wheat seedlings as a creation of nonproteinogenic storage form for the precursor for IAA biosynthesis when the growth was fast during germination. Nevertheless, free Try was essential but not the predominant amino acid in wheat seedlings (Figure 3). Thus, the role of other D-amino acids still remained obscure. We searched for other explanations for the possibility of using nonproteinogenic amino acids for wheat seedlings, which they possess in order to survive in ecologically unfavorable conditions.

Amino acids might be used in the formation of plant lectins or phytoagglutenines. Plant lectines may play the role of antibodies against soil bacteria and fungi and participate in the defense response of young seedlings because the localization of lectins was found in embryos and other parts of plant. The binding action of amino acids to a sugar moiety was provided by UDPG-transferase. UDPG-transferases are a widespread and abundant enzyme family with broad substrate specificity. As a model system, we used IAA as a substrate in order to evaluate the activity of UDPG-transferase in wheat shoots, because IAA is a derivative of the amino acid Try and closely related to it in indole and side chain structure (Table 4).

The activity of UDPG-transferase was high in all parts of the wheat plant. Therefore, wheat seedlings have a highly active system for balancing the IAA level that was produced by rapid synthesis from D-Try. As a whole, the IAA biosynthesis and its metabolism is sufficiently intense to provide for the fast growth of etiolated seedlings during the heterotrophic period in order to emerge from the soil and initiate photosynthesis. The D-amino acids, which are not involved in protein biosynthesis, might participate in the protection of young seedlings from pathogens, bacteria, and fungi by this very unique manner of joining with glucose or another sugar moiety. This objective will be the subject of following experiments.

References.

• Bell EA. 1980. Non-protein amino acids in plants. In: Enc Plant Physiol 8:403-432.
• Davies JS. 1977. Occurrence and biosynthesis of D-amino acids. In: Chemistry and biochemistry of D-amino acids 4:1-27.
• Kowalczyk S and Bandurski RS. 1991. Enzymic synthesis of 1-O-(indol-3-ylacetyl)-b-D-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme. Biochem J 279:509-514.
• Manabe H. 1986. Effect of exogenous D-alanine on D-alanyl-D-alanine content in leaf blades of Oryza australiensis Domin. Plant Cell Physiol 27:573-576.
• Manabe H and Ohira K. 1983. Effect of light irradiation on the D-alanylglycine content in rice leaf blades. Plant Cell Physiol 24:1137-1142.
• Ogawa T, Fukuda M, and Kei S. 1973. Occurrence of N-malonyl-D-tryptohan in pea seedlings. BBA 297:60-69.
• Ogawa T, Kimoto M, and Sasaoka K. 1977. Identification of D-aspartic acid and D-glutamic acid in pea seedlings. Agric Biol Chem 41:1811-1812.
• Rekoslavskaya NI. 1986. Possible role of N-malonyl-D-tryptophan as an auxin precursor. Biol Plantarum 28:62-67.
• Rekoslavskaya NI, Markova TA, and Gamburg KZ. 1988. Appearance of N-malonyl-D-tryptophan in excised leaves during wilting. J Plant Physiol 132:86-89.
• Rekoslavskaya NI, Yurieva OV, Shainyan BA, Kopytina TV, and Salyaev RK. 2002. Wheat racemase and the role of stereoisomers of N-malonyltryptophan during seed germination. Ann Wheat Newslet 48:141-143.
• Vicario PP, Green BG, and Katzen HM. 1987. A single assay for simultaneously testing effectors of alanine racemase and/or D-alanine. J Antibiot 40:209-216.

Changes in the aquaporin content in winter wheat membranes after deadaptation. [p. 118-119]

G.B. Borovskii, A.Yu. Yakovlev, S.V. Vladimirova, and V.K. Voinikov.

In the past decade, we have discovered that water transport in cells is not directly through membranes but through numerous channels in the membranes. These channels are formed by proteins adhering to aquaporins. Aquaporins are found in the plasma and vacuolar membranes in animal and plant cells (Maurel 1997; Connolly et al. 1998). By regulating the degree of aquaporin phosphorylation, the cell controls the permeability of a membrane to water (Maurel et al. 1997; Kjellbom et al. 1999) and changes in the amount of these proteins shift the range of regulation. During adaptation to low temperature, membrane permeability increases and water migrates into the intercellular spaces during freezing (Alberdi and Corcuera 1991). This increase in permeability very likely is associated with an increase of aquaporins in the membranes. We expect the reverse during deadaptation in the spring. To date, changes in the quantity of water-channel proteins during deadaptation of overwintered plants has not been investigated.

Materials and methods. The crowns and leaves of winter wheat plants of the cultivar Irkutskaia ozimaia were used in this study. This genotype is winter hardy and highly productive under the severe climatic conditions of eastern Siberia (Borovskii et al. 2001). Crowns, leaves, and soil monoliths with plants were sampled in the field in January. Crowns and leaves were used for membrane-fraction isolation. The remaining plants in the monoliths were left at room temperature for 1 month under natural illumination for de-adaptation. After 1 month, the crowns and leaves were harvested and the membrane fraction isolated. We identified aquaporins inside the microsomal membrane fraction, because antibodies demonstrated a high degree of specificity (Figure 4).

Wheat membranes were isolated by centrifugation at 105,000 g for 1 h. Proteins were dissolved in a sample-loading buffer at 65 C. Proteins were separated by SDS-PAGE using a mini-Protean II PAGE cell (Bio-Rad, U.S.A.) according to the manufacturer's instructions. Western blotting and immunodetection were as described by Timmons and Dunbar (1990) using anti-PIP (plasmalemma-intrinsic protein) and anti-TIP (tonoplast-intrinsic protein) primary antibodies (1:1000 dilution), kindly provided by Dr. A. Schaeffner (Institute of Biochemical Plant Pathology, München, Germany) and Dr. C. Maurel (Institut des Sciences Végétales, Gif-sur-Yvette, France), respectively.

Results and discussion. We observed a decrease in aquaporins in both leaves and crowns after deadaptation of winter wheat (Figure 5). Plasmalemma and tonoplast aquaporins decreased. This data supports the hypothesis that decreases in membrane water permeability occur after spring deadaptation. We assume that the permeability of membrane to water decreases in plants, because permeability is associated closely with freezing resistance. Alternatively, changes in the aquaporin content of the membrane could be connected with the start of the next stage plant development after overwintering plants reinitiate growth.

The aquaporin content culminates after development in the autumn; water exits the cell during freezing. We know that some aquaporins are strongly induced by ABA (Kaldenhoff and Eckert 1999). This fact indirectly confirmed our results, because ABA content is high during winter adaptation and decreases under deadaptation in the spring. Activation of the water channels is useful to expel water and entrance inside under extreme thawing. In our opinion, regulating the action of water channels under the freezing in the external spaces of the cell is the same mechanisms that takes place under the water stress (Kjellbom et al. 1999), by stress-increasing of Ca^2+^ content in the cytoplasm. After winter, a high aquaporin content is dangerous because Ca^2+^ content in the cytoplasm increases under any stress.

Changes in the permeability of cell membranes to water are very important for plant adaptation to freezing. The importance requires a tight control of permeability. Our results suggest that aquaporins are involved in adaptation of wheat to winter and deadaptaion in spring.

Acknowledgments. The work has been supported by the Russian Foundation of Basic Research (projects 02-04-48728 and 02-04-48599). We sincerely thank Dr. A. Schaeffner and Dr. C. Maurel for gift of antibodies.

References.

• Alberdi M and Corcuera LJ. 1991. Cold acclimation in plants. Phytochem 30:3177-3184.
• Borovskii GB, Stupnikova IV, Peshkova AA, Dorofeev NV, and Voinikov VK. 2001. Ann Wheat Newslet 47:179-185.
• Connolly DL, Shanahan CM, and Weissberg PL. 1998. The aquaporins. A family of water channel proteins. Internat J Biochem Cell Biol 30:169-172.
• Kaldenhoff R and Eckert M. 1999. Features and function of plant aquaporins. J Photochem Photobiol B Biol 52:1-6.
• Kjellbom P, Larsson C, Johansson I, Karlsson M, and Johansson U. 1999. Trends Plant Sci 4:308-314.
• Maurel C, Kado RT, Guern J, and Chrispeels MJ. 1995. Phosphorylation regulates the water channel activity of the seed-specific aquaporins a-TIP. EMBO J 14:3028-3035.
• Maurel C. 1997. Aquaporins and water permeability of plant membranes. Ann Rev Plant Physiol Plant Mol Biol 48:399-429.
• Timmons TM and Dunbar BS. 1990. Protein blotting and immunodetection. Methods Enzymol 182:679-688.
  

Using urea nitrogen for the nutrition of spring wheat under adverse temperatures. [p. 119-124]

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

Introduction. Urea is used widely in agriculture and is highly competitive with, and under certain conditions superior to, mineral forms of N fertilizers in its effect on yield and quality. For example, urea contributes to a greater accumulation of protein, gluten, and indispensable amino acids in wheat grain and other cereals during grain formation and maturation (Finney et al. 1957; Pavlov 1967; Schlehuber and Tacker 1967; Slukhai and Zrazhevsky 1971; Mitrofanov et al. 1973; Fox et al. 1986). Urea is taken up rapidly and metabolized by plants (Mokronosov et al. 1966; Pavlov 1967; Andrews et al. 1984). Urea increases the permeability of membranes and tissues and enhances the uptake, transferal, and reutilization of nutrients in plants (Mitrofanov et al. 1973; Turley and Ching 1986).

The mechanisms by which ammonium fertilizer and urea nitrogen affect plant metabolism are different (Tishenko et al. 1991). Thus, the role of urea as a N fertilizer has been studied in relatively sufficient detail, but the influence of adverse environmental factors on plant nutrition and physiology by this form of nitrogen have not. Over the last decade, researchers have had a great interest in studying the physiological response of plants to the nitrate and ammonium forms of N under stress conditions of salinity, low temperature, drought, and inadequate illumination (Chandra et al. 1986; Hubick 1990; Leidi et al. 1991; Gruz et al. 1993; Glyanko 1995).

Our results are derived from studying physiology of nutrition with urea nitrogen when spring wheat plants were exposed to a late spring frost (-6, -7 C) and low soil temperature (> 0 C) to compared to using the mineral forms of nitrogen.

Material and methods. Plant material and growth conditions. Soft spring wheat plants of the cultivar Skala were grown in containers (eight plants/container) in a growth chamber at the Siberian phytotron (Irkutsk, Russia) at a temperature of 19 ± 1 C/15 ± 1 C (day/night), illuminated by DRL-700 incandescent lamps. The light intensity was 14 ± 0.5 kLx with a 16-hour daylength. Infrared radiation from the lamps was suppressed by a water screen. The plants were grown using a sand­soil mixture with a small amount of total nitrogen (0.009 %). Macro- and microlements were supplied at half the normal rate into enameled containers filled with dry soil (Grodzinsky and Grodzinsky 1973). Watering was by weight with distilled water up to 70 % of the moisture capacity of the soil. To guard against any nitrification of the ammonium, the nitrification inhibitor 2-chlor-6-trichlormetyl pyridine (N-serve) was introduced into the containers at 1 % of the N dose.

Conditions of the artificial frost. A spring frost condition between -6 and -7 C was produced in a refrigerating chamber of the phytotron once the plants had reached the three-leaf stage. The chamber was not illuminated during the frost period. Temperature in the chamber was controlled automatically under a preset program (Kurets 1974). The program provided for a gradual decrease in temperature within the chamber from the optimum temperature (19 ± 1 C) to 0 C (at the rate of 1 C/12 min), followed by a decrease to the minimum subzero temperature (-6 and -7 C) at the rate of 1 C/22 min. After a 1.5-hour exposure to temperatures between -6 and -7 C, the temperature was raised to 0 C at the rate of 1 C/12 min. The temperature was raised from 0 C to the optimum temperature at the same rate. The total time of exposure of the plants to subzero temperature was 6 hr, of which 1.5 hour corresponds to the minimum subzero temperature. The relative air humidity within the chamber was 85-90 % during the frost. The containers with plants were placed in holes in plastic foam to avoid freezing the soil during the frost. One and one-half hours after the end of the frost (the temperature in the chamber was raised to 19 C), both control and experimental plants were fed through their roots with a mixture of three forms of N, one of which contained labeled 15N. The extra nutrition schemes were variant I, 15NH414NO3 + 14N - urea; variant II, 14NH415NO3 + 14N - urea; and variant III, 14NH414NO3 + 15N - urea.

In variant I, where the label was in the NH4 group, 25.9 mg 15N were introduced in each container and the enrichment of 15NH4NO3 was 95.31 weight percent of 15N; in variant II, 24.3 mg 15N with an enrichment of NH415NO3 of 89.66 weight percent of 15N; in variant III, 52.3 mg 15N were introduced with a urea enrichment of 93.84 weight percent of 15N. The total amount of nitrogen that was introduced into the vessels during the extra nutrition was 106.4 mg in the first two variants and 101.3 mg in variant III.

Soil temperature reduction. To reduce the temperature in the root zone, containers with plants were placed in thermal chambers through which water at 5 ± 1 C and 19 ± 1 C was passed, maintaining the required soil temperature (Kurets 1974).

Chemical analyses. Protein in the triturated leaves was precipitated with trichloroacetic acid. Nucleic acids and other soluble compounds were removed from the protein precipitate (Klyachko et al. 1971). The protein was digested in concentrated sulfuric acid with a catalyst, selenium (Se). Protein nitrogen was distilled by the micro-Kjeldale method and determined by the titrimetric method of Ermakov et al. (1987). Samples were analyzed for enrichment of 15N by means of a mass-spectrometer MI-1309. The content of labeled N in samples was determined by a formula for isotopic dilution (Korenkov 1977). The atomic percent of 15N was converted to weight percent of 15N (Korenkov 1977). The activities of glucose-6-phosphate dehydrogenase (G-6-PD) and malate dehydrogenase (MD) were determined using biochemical tests (Boehringer and Soehne GmbH Mannheim, Germany) in cell-free, unpurified root extracts. Urease activity was determined according to Bollard et al. (1968), and the protein in cell-free preparations was quantified according to Lowry et al. (1951). The biological and analytical repeatability of assays was fivefold and threefold, respectively. Results are represented as the arithmetic mean with a standard error. The confidence level of the differences was evaluated by the Student t-test (tst). Least significant difference for comparing treatment means at the 0.95 probability level.

Results and discussion. Effect of late spring frost. Of 195 plants that underwent frosts, 64 (32 %) had one damaged leaf, eight had two damaged leaves, and three plants died. Thus, 38 % of the plants showed visually observable damages.

The sample for quantifying protein was made from the laminas of two plants having no visible damage. The plants did not show any substantial differences in protein accumulation in their leaves during the first 9 days after the frost, the absolute content in both control and experimental plants increased by a factor of 1.6 to 1.7. Labeled N is incorporated into leaf protein at a different rate depending on the form of N-fertilizer (Table 5). For example, 9 days after the frost, 552.9 µg 15N from urea, 137.0 µg from the ammonium group, and 73.8 µg from the nitrate group were determined in the protein of the control plants. The percentage of labeled N utilization by the plants from fertilizers amounted to 1.06, 0.53, and 0.30, for urea, ammonium, and nitrate, respectively. During frost, this remains regular (Table 5). The difference is that a short exposure to subzero temperature promotes the incorporation into protein of the label from urea. When compared to control plants, the label incorporation is 115 and 150 % at 3 and 9 days after the frost, respectively (differences at td>tst).

Label incorporation into protein 1, 3, and 9 days after the frost also is stimulated from the 15NO3 group. The confidence level of the differences between the control and the assays are very high (P > 0.99). With regard to the effect of frost on the incorporation of the label from the 15NH4 group, a reliable decrease in 15N incorporation into protein on day 3 is observed (P > 0.95), whereas the differences are unreliable at 1 and 9 days after the frost (td<tst).

The utilization of labeled N from different forms of N on day 9 after the frost was 1.58, 0.57 and 0.48 %, from urea, ammonium, and nitrate, respectively. Thus spring wheat seedlings predominantly utilize urea N in synthesizing the protein. Temperature stress has a stimulating effect on this process. The control and experimental plants did not differ in absolute protein N content in the leaves (Table 5), suggesting that, during increased catabolic processes such as after frost, plants are able to shift the state of decay-synthesis of proteins toward the latter through an intense utilization of urea N.

The predominant utilization of urea from the mixture of three forms of N can probably be explained by a couple of factors. First, the relatively easy uptake of urea by roots. Second, the fast transport of urea (or its products) to aerial organs and subsequent use in metabolism.

In comparison with mineral forms of N (NO3^-^ and NH^4+^), the mechanism of urea uptake by plants is not yet understood (Van Beusichem and Neeteson 1982). We anticipate that urea, as a neutral compound, is taken up by root cells with a minimum expenditure of energy and a high proportion is transported to aerial organs in an unchanged form. Urease activity in wheat roots and seedling leaves when the plant roots receive extra nutrition of urea provides some evidence. Activity of urease in leaves increases by a factor of 2.9, whereas enzyme activity in the roots is uncertain.

Chen and Ching (1988) induced leaf urease activity when barley plants are sprayed with urea solution. They detected urease isozymes, which were synthesized only during the period of an abrupt increase in enzyme activity. Our data indicate that spring wheat seedlings contain a sufficiently active constitutive form of urease in their roots and a less active form in leaves (medium without N). Under the influence of extra nutrition of plant roots with urea, urease activity changes little in roots but increases abruptly in leaves. The latter is likely to be associated with the de novo synthesis of enzyme.

The reasons for stimulating the uptake of label from urea as an effect of frost are unclear. We determined the urease and nitrate reductase activity in wheat leaves as an effect of the frost (within 1 and 3 days) and found that the activity of both enzymes was enhanced. However, we only can explain the presence in cells of a sufficient number of NH4+ ions needed for the synthesis of amino acids. The mechanism of the effect of low temperature on the transcription-translation apparatus in leaves when plants are fed with different forms of nitrogen remains to be elucidated.

Reduced temperature effect of soil. Urea as fertilizer behaves in a peculiar fashion at low above-freezing temperatures in the root zone. We found that after exposure to low temperature (5 ± 1 C), G-6-PD and MD activity increases in roots by a significantly greater amount when the plants were fed with urea as compared to NO3^­^ and NH^4+^. The activity of G-6-PD in the roots by urea is stimulated by 7-fold, as opposed to 3.2- and 3.9-fold for the NO3^-^ and NH^4+^ N-sources, respectively. Under normal temperatures, enzyme activity in plants is higher with NO3 nutrition. The stimulating effect of NO3 on enzymes of the pentose monophosphate pathway of carbohydrate oxidation has been reported (Givan 1979). The activity of MD at near-freezing temperature increases in roots by 267, 167, and 136 % in variants with urea, NH^4+^, and NO3­, respectively. At the optimum temperature in the root zone (19 ± 1 C), however, the activity of these enzymes during urea nutrition of plants is lower when compared to variants with other nitrogen forms. A possible mechanism to explaining the stimulation of the G-6-PD and MD activity under stress could be the dissociation of the multidimensional forms of enzymes into simpler subunits having increased activity. The presence of electrophoretically different forms of enzymes suggests that under different conditions in the medium the relationship of different molecular forms of enzymes can change drastically (Petrova et al. 1985), which is responsible for the increase or decrease in enzyme activity.

We observed a greater stimulating of enzyme activity under low-temperature effect in the presence of urea. In protein chemistry, urea is known as a dissociating agent of proteins (Zolkiewski et al. 1995). At low temperatures, conditions that allow the penetration of urea to places where compartmentalizing of enzymes may be created in cells and the molar concentration suffices to have a dissociating effect on enzymes. An alternative explanation for the activation of the G-6-PD and MD enzymes could be an enhancement, at low temperature, of other processes such as anaplerotic pathways for the assimilation of carbonic acid during the enzymatic decomposition of urea in plant cells. This pathway involves enhancing the carboxylation processes with the participation of root phosphoenolpyruvate carboxylase and other CO2-fixing enzymes resulting in products that are used in the Krebs cycle.

When urea is used to nourish plants in the root zone at low temperature, root growth is enhanced. According to our data from a water-culture assay, the presence of urea as the only growth source in the nutrient solution causes enhanced growth of plants if the temperature in the root zone was 5 ± 1 C. This effect of urea on root growth was not observed in the root zone at the optimum temperature. This assay was repeated in soil-cultured plants. In this case, nitrogen in the form of different fertilizers was introduced at 42 mg/kg soil (210 mg/container). All other elements were introduced at one-half the normal concentration. Once seedlings appeared, containers with seedlings were placed in different temperature conditions and the plants were grown until the third leaf appeared. At optimum soil temperature, the plants reached the 3-leaf stage within 13-14 days; at low temperature this occurred with in 21-23 days.

Our results showed that at low soil temperature and optimum air temperature (19 ± 1 C), the root dry weight in the variant with urea was higher when compared to plants grown with the other forms of N. The mean length of roots in the variant with urea at both the low and optimum temperatures was greater when compared with the other N-sources (Table 6). The root wet weight during urea nutrition under low temperature conditions in both water and soil culture approaches or exceeds that in the variant without N. Nitrogen deficiency and phosphorus in the medium is known to promote growth of the plant root system (Barber 1979), and the presence of these elements leads to a decrease in intensity of growth. In this case, during urea nutrition under low soil temperature conditions, plant roots behave as in the variant without N.

The mechanism responsible for enhancing root growth in the absence of N (or phosphorus) in the medium is unknown. Barber (1979) suggests that a stem-connected feedback mechanism causes an increase in root growth. Such a mechanism could be a hormonal imbalance in wheat roots during nutrition of plants with urea and other forms of N. According to our data, the relation between indoleacetic acid and abscisic acid in root tissues of wheat seedlings varies according to the form of N and soil temperature (Glyanko 1995). Lips (1997) also reported that variation in the balance between abscisic acid and cytokinins in roots during nitrate and ammonium nutrition has an effect on the growth of roots and aerial organs and contributes to adaptation of plants to stress effects (salinization or moisture deficiency). Thus, enhancement of root growth in conditions of near-freezing temperatures is effected under the influence of urea, and activation of urea N in protein molecules as an effect of frost is manifested by the adaptive and reparative changes in wheat plants induced by the form of N.

References.

• Andrews RK, Blakeley RL, and Zerner B. 1984. Urea and urease. In: Advances in Inorganic Biochemistry (Eichhora GL and Marzilli LG eds), Elsevier, New York. 6:245-283.
• Barber SA. 1979. Uptake of nutrients from soil by plant roots. Physiol Biochem Cultiv Plants 11:209-217 (in Russian).
• Bollard EJ, Cook AK, and Turner NA. 1968. Urea as sole source of nitrogen for plant growth. 1. The development of urease activity in Spirodella eligorriza. Planta 83:1-12.
• Chandra R, Radhuver P, and Sirohi GS. 1986. Influence of moisture stress and nitrogen on growth and yield of pea and sorghum. Ann Arid Zone 25: 225-231.
• Chen Y and Ching TM. 1988. Induction of barley leaf urease. Plant Physiol 86:941-945.
• Cruz C, Lips SH, and Martins-Loucao MA. 1993. Effect of root temperature on carob growth: nitrate versus ammonium nutrition. J Plant Nutrit 16:1517-1530.
• Ermakov AI, Arasimovich VV, and Yarosh NP. 1987. Methods of biochemical investigation of plants. VO Agropromizdat, Leningrad Pp. 237-238 (in Russian).
• Finney KF, Mayer JM, Smith FM, and Fryer HC. 1957. Effect of Pawnee wheat with urea solution on yield, protein content and protein quality. Agron J 49:341-347.
• Fox RH, Kern JM, and Piekieler WP. 1986. Nitrogen fertilizer source, and method and time of application effects on no-till corn yields and nitrogen uptakes. Agron J 78:741-746.
• Givan CV. 1979. Metabolic detoxification of ammonia in tissues of higher plants. Phytochem 18:373-383.
• Glyanko AK. 1995. Nitrogen nutrition of wheat at low temperatures. Nauka, Novosibirsk (in Russian).
• Grodzinsky AM and Grodzinsky DM. 1973. Concise manual on plant physiology. Naukova Dumka, Kiev (in Russian).
• Hubick KT. 1990. Effects of nitrogen source and water limitation on growth, transpiration efficiency and carbon-isotope discrimination in peanut cultivars. Aust J Plant Physiol 17:1413-1430.
• Kurets VK. 1974. The Irkutsk phytotron. Nauka, Novosibirsk (in Russian).
• Klyachko N, Yakovleva LA, and Kulaeva ON. 1971. Change in protein synthesis in the cotyledons of pumpkin in connection with their age. Fiziologiya Rasteniy (Sov Plant Physiol) 18:1225-1231.
• Korenkov DA. 1977. Methods of application of nitrogen isotope 15N in agricultural chemistry. Kolos Publishing House, Moscow (in Russian).
• Leidi EO, Soares MIM, Silberbush M, and Lips SH. 1991. Salinity and nitrogen nutrition studies on peanut and cotton plants. J Plant Nutrit 15:591-604.
• Lips SH. 1997. The role of inorganic nitrogen ions in plant adaptation processes. Rus J Plant Physiol 44:421-431.
• Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.
• Mitrofanov BA, Okanenko AS, and Pochinok KhN. 1973. Effect of urea spray application of winter wheat on intensity of photosynthesis and grain quality. Physiol Biochem Cultiv Plants 5:232-238 (in Russian).
• Mokronosov AT, Ilinych ZG, and Shukolyukova NI. 1966. Assimilation of urea by potato plants. Fiziologiya Rasteniy (Sov Plant Physiol) 13:798-806.
• Pavlov A. 1967. Protein accumulation in wheat and maize grains. Nauka, Moscow (in Russian).
• Petrova OV, Kolosha OI, Mishustina PS, and Sukhareva IB. 1985. Enzyme form multiplicity and its modification in winter wheat in the period of adaptation to low temperatures. Physiol Biochem Cultiv Plants 17:361-366 (in Russian).
• Schlehuber AM and Taker BB. 1967. The growing of wheat. In: Wheat and wheat improvement (Reitz LP and Quisenberry KS eds). American Society of Agronomy, Madison, WI. Pp. 140-198.
• Slukhai SI and Zrazhevsky MN. 1971. Increase of winter wheat grain quality under condition of irrigation. Physiol Biochem Cult Plants 3:303-313 (in Russian).
• Tishenko NN, Nikitin DB, Magomedov IM, and Moran E. 1991. Effect of nitrate and ammonium forms of nitrogen fertilizers on sugarcane photosynthesis and growth parameters. Physiol Biochem Cultiv Plants 23:446-452 (in Russian).
• Turley RH and Ching TM. Physiological responses of barley leaves to foliar applied urea-ammonium nitrate. Crop Sci 26:987-993.
• Van Beusichem ML and Neeteson JJ. 1982. Urea nutrition of young maize and sugarbeet plants with emphasis on ionic balance and vascular transport of nitrognous compounds. Neth J Agric Sci 30:317-330.
• Zolkiewski M, Nosworthy NJ, and Ginsburg A. 1995. Urea-induced dissociation and unfolding of dodecametric glutamine synthetase from Escherichia coli ­ calorimetric and spectral tudies. Protein Sci 4:1544-1552.

VAVILOV INSTITUTE OF GENERAL GENETICS, RUSSIAN ACADEMY OF SCIENCES
Gubkin str. 3, 119991 Moscow, Russian Federation.

SHEMYAKIN AND OVCHINNIKOV INSTITUTE OF BIOORGANIC CHEMISTRY, RUSSIAN ACADEMY OF SCIENCES2
Ul. Miklukho-Maklaya 16/10, Moscow, Russian Federation.

Isolation and characterization of antimicrobial peptides from Triticum kiharae. [p. 124-125]

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

All living organisms have evolved mechanisms with which to defend themselves against pathogen attack. This innate immunity involving the production of antimicrobial peptides is one of the most ancient and widespread defense strategies. After defense peptides are produced by transcription and translation of a single gene, they can be delivered rapidly after infection with a limited input of energy and biomass and display differential activity against different types of microorganisms (Thomma et al. 2002). Different families of antimicrobial peptides have been identified, including thionins, defensins, lipid-transfer proteins (LTPs), hevein-type peptides, and knottin-type peptides.

We hoped to identify the antimicrobial peptides in T. kiharae, which is highly resistant to most pathogens infecting cultivated wheat. T. kiharae has been used in our laboratory in crosses to generate lines resistant to such fungal pathogens as powdery mildew and brown rust.

Materials and methods. The peptide fraction was extracted from T. kiharae flour with 10 % acetic acid (flour to solution ratio of 1:10) for 1 h at room temperature. The supernatant was lyophilized and subjected to chromatography. The acid-soluble fraction was separated by gel-exclusion chromatography on a Sephacryl S-100 HR column using 10 % acetonitrile containing 0.1 % TCA as eluent. The chromatographic fractions were tested for the antifungal activity against several fungi (Helminthosporium sativum, Alternaria consortiale, Rhizoctonia solani, Botritis cinerea, and Fusarium culmorum). The active fraction, which caused inhibition of fungal growth and morphological changes, was separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC-fractions were tested against fungi and characterized by mass spectrometry (MS) and sequencing.

Results and discussion. Separation of acid-soluble peptides on a Sephacryl column produced six fractions designated from A to G. Only fraction D exhibited antifungal activity against most fungi assayed. This fraction was further separated by RP-HPLC. Several fractions were obtained. Their molecular masses were measured by MS, and N-terminal sequences identified by automatic sequencing. The peptide masses separated by RP-HPLC are in Table 1.

The N-terminal sequences of all fractions were determined. Two fractions were identified: Fr. 4: AXQASQLAVXASAILGGTKPSGE and Fr. 5: KSXXK/RSTL

The N-terminal sequence of fraction 4 coincides with that of LTP; however, three substitutions at positions 3, 4, and 5 have been observed (Garcia-Olmedo et al. 1998). Plant LTPs are 90-95 amino acid polypeptides that have been identified (at a protein and/or cDNA levels) in various tissues from a high number of mono- and dicotyledonous species. They were found to be distributed throughout the plant. Antimicrobial activity of LTPs has been reported for all members of the family tested. The relative activities of different LTPs vary between pathogens, suggesting that they have some degree of specificity. The mass of LTP from T. kiharae is lower than that described in the literature for other members of this family.

According to the N-terminal sequence, fraction 5 corresponds to a/b purothionins. The toxicity of thionins to plant pathogens is known from investigations into the susceptibility to wheat endosperm thionins of phytopathogenic bacteria in the genera Pseudomonas, Xanthomonas, Agrobacterium, Erwinia, and Corynebacterium. Purified genetic variants of these thionins differed in activity and showed some degree of specificity. Recent experiments in planta also are indicative of a defense role for the thionins.

Other fractions obtained by RP-HPLC of T. kiharae peptides were heterogeneous; therefore, their sequencing produced inconclusive results. Some low-molecular peptides were sequenced directly after the separation of the total acetic-acid extract on an RP-HPLC column. The sequences obtained were TRQLSLRG and TRQLSPRG. Homologous proteins were not found in the data banks, so their functions remain unknown.

These results indicate that T. kiharae possesses different types of antimicrobial peptides.

References.

• Thomma B, Cammue B, and Thevissen K. 2002. Plant defensins. Planta 216:193-202.
• Garcia-Olmedo B, Molina A, Alamillo J, and Rodriguez-Palenzuela P. 1998. Plant defense peptides. Biopolymers (Peptide Science) 47:479-491.

Distribution of hybrid necrosis genes in common wheat cultivars of Australia. [p. 125-127]

V.A. Pukhalskiy, S.P. Martynov, and E.N. Bilinskaya.

We studied the necrosis genes in modern cultivars of spring common wheat of Australia. The distribution of hybrid necrosis genes in the old local cultivars was first investigated by Tsunewaki and Hori (1967, 1968), who showed that the Ne1 ne2 and ne1 ne2 genotypes prevailed by the end of the 19th and early in the 20th centuries. The available data on the wheat cultivars of Australia and Oceania indicate that 25.4 % are of the Ne1 ne2 genotype, 18.9 % are ne1 Ne2, and 55.7 % are ne1 ne2 Pukhalskiy( et al. 2000). This genotype distribution resulted from nearly a century of breeding in Australia. We thought it interesting to investigate this parameter at the end of the 20th century.

Materials and methods. The necrotic genotype was analyzed in 48 Australian cultivars of spring common wheat. The spring common wheat cultivars Marquillo (Ne1sNe1s ne2ne2 genotype) and Balaganka (ne1ne1 Ne2sNe2s) were used as testers. Crosses were conducted under field conditions by standard procedures including emasculation and isolation of spikes. The F1 and F2 hybrids were grown in the field. Hybrid necrosis traits were evaluated at different growth stages.

Results and discussion. The distribution of the different necrosis genotypes in Australian wheat cultivars shows that breeding led to complete elimination of the Ne1ne2 genotype (Table 2). If we estimate the ratios of necrotic genotypes in all 46 cultivars (except for cultivars Beulah and Bt-Schomburgk where the presence of the Ne2 gene is problematic), the results are as follows: 76.1 % of cultivars possess the ne1 ne2 genotype and the ne1 Ne2 genotype is found in 23.9 % of cultivars.

The ratios for wheats at the beginning of the 20th century were different (Tsunewaki et al. 1967). Among 72 cultivars examined, the ne1 ne2 genotype was found in 57 (79.2 %) of the cultivars, Ne1 ne2 in 14 cultivars (19.4 %), and ne1 Ne2 (1.4 %) only in one cultivar. The ne1 Ne2 genotype was found in the cultivar Atlas (Tsunewaki et al. 1968). The authors did not indicate whether Atlas is a winter or a spring cultivar. In all probability, Atlas was one of the two winter wheat cultivars studied.

We suppose that the observed changes in the distribution of hybrid necrosis genes were due to the Green Revolution and to the wide use of CIMMYT material in the Australian breeding programs.

Pedigree analysis of the Australian wheats using the GRIS 3.5 (Martynov and Dobrotvorskaya 1993) shows the Brazilian landrace Turco as the source of the Ne2 gene. In addition, this gene could be derived from the Argentinian landrace Barleta or the Japanese cultivar Norin 10, the donor of the short-stem trait, which has the Ne2 gene from the landrace Mediterranean through the old, American cultivars Lancaster and Fultz.

Acknowledgment. The authors are grateful to Michael MacKay, the curator of the Australian collection of winter cereals, for the seeds of modern Australian wheat cultivars used in this study.

References.

• Martynov SP and Dobrotvotvorskaya TV. 1993. Breeding-oriented database on genetical resources of wheat. Ann Wheat Newslet 39:214-221.
• Pukhalskiy VA, Martynov SP, and Dobrotvorskaya TV. 2000. Analysis of geographical and breeding-related distribution of hybrid necrosis genes in bread wheat (Triticum aestivum L.). Euphytica 114:233-240.
• Tsunewaki K and Hori T. 1967. Distribution of necrosis genes in wheat. IV. Common wheat from Australia, Tibet and Northern Europe. Jap J Genet 42:245-250.
• Tsunewaki K and Hori T. 1968. Necrosis genes in common wheat varieties from Australia, Tibet and Northern Europe. Wheat Inf Serv 26:22-27.
  

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

Genealogical analysis of Russian and Ukrainian winter wheat resistant to common bunt. [p. 127-133]

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

Common bunt is one of most serious diseases of bread wheat. This disease is distributed in many regions of the Russian Federation including the Northern Caucasus, Central Black Soil region, Volga region, and Non-Black Soil zone. Resistance to common bunt in winter wheat was measured by comparing groups of resistant and susceptible cultivars from the Russian Federation and Ukraine using a genealogical approach.

Data on winter bread wheat cultivars were taken from the database GRIS 3.5 of the Information and Analytical System of Wheat Genetic Resources (Martynov and Dobrotvorskaya 2000). A set of 199 cultivars with known resistance/susceptibility to common bunt and known pedigrees were divided into resistant (Table 1) and susceptible (Table 2) groups.

Tracing expanded pedigrees with the aid of the GRIS program has established the probable donors and sources of resistance to common bunt (Table 1). Except for eight cultivars for which it was impossible to identify the source of resistance, the source of resistance to common bunt 36 cultivars (82 %) was from local sources mainly A. glaucum via PPG-599, Crimean, Odessa local cultivar (LV-Odessa) via Zemka, Eliseevskaya rye, and Yaroslav emmer. Other cultivars (18 %) received resistance genes both from local and foreign sources; Florence (Bt3) and Oro (Bt4, Bt7). A number of cultivars have ambiguous estimations of resistance to bunt (marked by an * in Table 1). For example, Bezenchukskaya 380 is considered resistant in the Lower Volga region but susceptible in other areas. Moskovskaya 70 and 642, Moskovskaya nizkostebelnaya, Chaika, and Yantarnaya 50 are classified as resistant, but data from State Varietal Trials indicates susceptibility. Skorospelka 1 and 3, from source data, and Odesskaya 12, from State Varietal Trials, are resistant, but data from the Vavilov Institute identifies them as susceptible. We assume that the conflicting data are a consequence of the different race compositions of local pathogen populations. Krivchenko (1984) has identified 37 different pathogen races. Analyzing the geographical distribution of the pathogen races, we identified two groups appropriate to two conventional regions; north and south of latitude 49 N. Races 1, 9, 15, 17, and 20 comprised the southern group and 2, 14, 16, 25, 31, 34, and 37 were specific to the northern group. Races 6 and 11 were common to both groups. We assume that the sources of resistance differ in southern and northern regions. Therefore, we analyzed groups of resistant and susceptible cultivars divided into southern and northern subgroups (see Table 1 and Table 2). Among the cultivars of the southern area, the basic sources of resistance are the Odessa local variety (LV-Odessa) via Zemka, selection from Crimean (CI-1435), and foreign sources via Brevor and CIMMYT cultivars. In the northern subgroup, the number of sources of resistance is more limited; A. glaucum via PPG 599 and Eliseevskaya rye.

In a three-way ANOVA of the matrixes of ancestor contribution (Table 3), we investigated the resistance (factor A) with two classes (resistance and susceptibility), the region of origin (factor B) with two classes (south and north), and the original ancestor or hypothetical source of resistance (factor C) with the number of classes (c = 11). The analyzed sample included 52 resistant cultivars (including 23 from the southern and 29 from the northern regions) and 147 susceptible cultivars (including 88 from the southern and 59 from the northern regions). The data were transformed through arcsines. The effects of all investigated factors and interactions, except for interaction (A x B) were highly significant. Highly significant interactions (A x C), (B x C), and (A x B x C) indicate specific differences between the distribution of the contributions of hypothetical sources of resistance in groups of resistant and susceptible cultivars occurring from various regions. Differences in the race composition of regional populations of pathogen explain this fact.

In the northern region, the contributions of A. glaucum and Eliseevskaya rye are higher in the group of resistant cultivars. In the southern region, the Odessa local variety prevails among resistant cultivars (Table 4). In the northern region, the contribution of LV-Odessa is higher in the group of susceptible cultivars, confirming the race specificity of this resistance source. Yaroslav emmer, in the northern region, and foreign sources (Oro, Florence, Federation, and T. timopheevii), in the south, are effective, although their contribution is not significant when compared with the group of susceptible cultivars.

This analysis shows that number of sources of a vertical resistance to bunt used in the winter wheat-breeding programs in the Russian Federation and Ukraine is not sufficient. The high number of genotypes with identical reaction to bunt causes genetic uniformity in the cultivars. The narrowing of the genetic diversity from a few identical genes can cause a change in the pathogen population and increase susceptibility on homogeneous genetic material.

Efficient horizontal (nonracespecific) resistance, which is shown as incomplete resistance to all races of a pathogen and in varying degrees suppresses its development, also depends on the genetic diversity of the released cultivars. A study of latent genetic diversity in winter wheat cultivars from the Russian Official List has shown that the overwhelming majority (96 %) of cultivars recommended for cultivation in the Russian Federation are the descendants of Bezostaya 1 and/or Mironovsakaya 808. In the Central Black Soil zone and the Northern Caucasus and Middle and Lower Volga regions, the genetic diversity is acceptable, whereas the Central Non-Black Soil and Volga-Vyatka regions of the Russian Federation are characterized by low genetic diversity. The majority of cultivars recommended for these regions are related at the full- and half-sib level.

A key problem of breeding for resistance to bunt is use of the new sources of resistance. In addition to the 11 known resistance genes (Bt1-Bt10 and BtZ), 11 new genes have now been identified. Ukrainian researchers have identified six new genes; Bt11 from Sel. M-6623, Bt12 and Bt13 from Lutescens 6028, and Bt14 from Erythrospermum 5221 (Novokharka et al. 1990) and Bt15 and Bt16 from Ferrugineum 220/85 (Babayants and Dubinia 1990). CIMMYT researchers have identified five new genes, which, unfortunately, have been given the same gene designations; Bt11 (from PI-554119), Bt12 (from PI-119333), Bt13 (from Thule III), Bt14 (from Doubbi), and Bt15 (from Carleton) (Wilcoxson and Saari 1996). In addition, two presumably new genes in lines Erythrospermum 60-89 and Ferrugineum 124-88 were identified (Babayants et al. 1999). Some parental forms of Erythrospermum 5221, Ferrugineum 220/85, Erythrospermum 60-89, and Ferrugineum 124-88 are unknown, which does not enable pedigree analysis. We could analyze the pedigree only of Lutescens 6028 and are now able to explain bunt resistance in this line.

Tracing the transmission of Bt-genes on the expanded pedigrees the has shown that Lutescens 6028 (Selection 101/Manella//Kavkaz) can have genes Bt1, Bt3, Bt4, Bt6, and Bt7 from Selection 101 (Figure 1) that has the following cultivars and genes in its pedigree: Rex (Bt1 and Bt7), Rio (Bt6), Oro (Bt4 and Bt7), Florence (Bt3), Burt (Bt1, Bt4, and Bt6), and Brevor (Bt1, Bt3, Bt4, and Bt6). Novokhatka et al. (1990) could not explain the results of segregation of resistance in crosses between 'Lutescens 6028/Bt4 (monogenic line)' and 'Lutescens 6028/(Bt6) Rio'. The first cross segregated 74:26, which corresponds to the theoretical ratio 189:67 (r^2^ = 0.002) suggesting four genes (one basic and three duplicate-complementary genes (Manjunath and Nadaf 1983). A segregation of 57:58 was found in the second cross, corresponding to a theoretical 121:135 (r^2^ = 0.24) and suggesting four genes (two basic complementary and two duplicate-complementary genes (Manjunath and Nadaf 1983). Thus, we cannot prove that the resistance genes in Lutescens 6028 are nonallelic and independent from previously described genes Bt1, Bt3, Bt4, Bt6, and Bt7. The high level of resistance in Lutescens 6028 may come from a combination of all these genes.

Our analysis was made on the basis from information about resistance or susceptibility of winter wheat received from different authors by different techniques with different combinations of races in local pathogen populations. Therefore, we consider the data on source of resistance and statistical estimations made by comparing samples of resistant and susceptible cultivars as preliminary. Nevertheless, based on genealogical information, the data will be useful in conditions of artificial inoculation with certain races of the pathogen and the use of a standard set of differentials.

References.

• Babayants LT and Dubinina LA. 1990. A novel donor of wheat resistance to bunt (Tilletia carries (DC) Tul., T. laevis Kuehn.) and its genetical background. Rus J Genet 26(12):2186-2190 (in Russian).
• Babayants LT, Dubinina LA, and Yushchenko GM. 1999. Genetical background of resistance to common bunt (Tilletia carries (DC) Tul.) for new wheat lines. Cytol Genet 33(6):25-30.
• Krivchenko VI. 1984. Resistance to bunt for grain crops. Moscow:Kolos. 304 pp. (in Russian).
• Manjunath A and Nadaf SK. 1983. A ready reckoner of expected F3 breeding behaviour useful in linkage studies. Madras Agric J 70(6):360-365.
• Martynov SP and Dobrotvorskaya TV. 2000. A study of genetic diversity in wheat using the Genetic Resources Information and Analytic System GRIS. Rus J Genet 36:195-202.
• Novokhatka VG, Mochalova LI, and Odintsova IG. 1990. New genes in wheat for resistance to common and dwarf bunt (Tilletia carries (DC) Tul., T. laevis Kuehn., T. controversa Kuehn.). Rus J Genet 26(10):1808-1814 (in Russian).
• Wilcoxson RD and Saari EE eds. 1996. Bunt and smut diseases of wheat: Concepts and methods of disease management. CIMMYT, Mexico. 66 pp.