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.
L.V. Dudareva, S.V. Lankevich, V.M. Sumtsova, E.G. Rudikovcka, and R.K. Salyaev.
The impact of laser radiation on living organisms, including plants, has been raising an intense interest from researchers since the moment the laser was invented in the mid 1960s. Nevertheless, up to now, not a single theory can explain all the effects that lasers make on living things because of the relative complexity of biological systems and the difficulty of analyzing the regularities of energy transformation in living tissues.
Of particular interest is the impact of low-intensity laser radiation on biological objects. This impact does not normally cause damage. Vice versa, the stimulating influence of laser radiation on many physiological processes, in humans, animals, and plants, is regarded as proven fact. Few studies have investigated any possible paths of the stimulating influence of low-intensity laser radiation on plants. However, from an evolutionary viewpoint, plants are well suited to the perception of light energy and its physiological use. The impact of light on plants is not only restricted to photosynthesis, and many other photobiological processes, such as photoregulation, should be noted. The physiological status of a plant depends, to a large extent, on light intensity, its spectral composition, radiation dose, and the period of illumination. Apart from the chloroplasts, plant tissues are rich in pigments that perform various, but primarily signaling, functions in plant cells. The study of the biological impact of low-intensity laser radiation on the plants may be aimed not only at the identification of optimal conditions for its practical application, but for the study of fundamental regularities of light impact on plant organisms.
The literature and our data prove that low-intensity laser radiation may produce a stimulating influence on various physiological processes, including those that do not show a pronounced interconnection. For example, the lack of an interaction between the degree of plant regeneration and callus formation in individual genotypes confirms a lack of connection between the genetic factors that determine these two characteristics (Yurkova 1989).
We have established that the primary response of plant tissue to radiation is an increase in the content of secondary products of peroxide oxidation. Our data demonstrate that laser light stimulates morphogenetic processes in plant tissues at later stages as well (Salyaev et al. 2001). We believe that this stimulation may be conditioned metabolic changes caused by the change of content of a number of compounds formed as a result of the primary photoreactions. Such compounds might also include products of peroxide oxidation, with an increase in their amount in response to the impact of laser radiation. This increase, in turn, affects membrane properties and changes its functional state. The impact of PLO on the phospholipid stratum of membranes is well studied and may be reduced to several principal effects (Vladimirov 1999); a selective increase in membrane permeability and a reduction of their electrical stability. One of the major results is an increase in the Ca2 + concentration inside the cells. The sequence of events following laser radiation exposure may be as follows:
Activation of intracellular processes. PLO-products accumulation may act as a signal not only for the start-up of relevant protection mechanisms, but, probably, for some secondary responses, perhaps, at the transcription level (0°C, 2001). This probability is indirectly confirmed by the stimulating effect of laser light on morphogenetic processes in wheat and wild crops tissue cultivar. When exposing plants to laser radiation, it is important to remember that is is possible to dose the stress impact strictly.
Based on data from the stimulation of morphogenetic processes in wheat tissue culture by low-intensity laser radiation (Salyaev et al. 2001), we suggest that the changes observed should be accompanied by molecular shifts and structural reconstruction in the tissues subjected to radiation. Chirkova (2000) indicated that such reconstructions should take place primarily in cell and organellar membranes. These reconstructions produce a profound impact on all forms of functional activity in the membrane, with lipids to a considerable extent. In lipids, fatty acids are the primary subject influenced by both genetic control and environmental conditions. Stress may cause shifts in the proportion between various groups of fatty acids, and the degree of their nonsaturation may change. The length of fatty acids chains, positional situation of double bonds, or the number of polar groups also may change. If we are able to show a difference in the structure of the lipid matrix in control tissues and tissues subjected to radiation, we could confirm the biological impact of laser light on plants via its influence on membrane structure. Thus, part of our work studied qualitative changes in lipid structure caused by the impact of low-intensity, laser radiation by infrared spectroscopy.
Callus tissue from the wheat cultivar Skala, a Siberian selection, were subjected to irradiation on the second day after the first transfer. The radiation dose (4.5 J/cm2) was identical to that in experiments investigating the impact of laser radiation on morphogenetic processes in wheat callus. Extracts from unradiated calli were used as control. Calli were taken for analysis 72 hours (3 days) after irradiation. The molecular spectra obtained found that coherent radiation caused significant changes in the structure of lipids (Table 1).
| Spectrum region (per cm) | Control callus | Test callus |
|---|---|---|
| 3,300 | Significant reduction of OH-2 and NH- groups | |
| 1,7301,690 | Carbonyl (C=O) fluctuations, intense bands | Indistinct bands |
| 1,6201,630 | Aromatic groups, most likely phenols | Absence of bands |
| 1,2301,050 | Phosphate groups, symmetrical and asymmetrical PO-2 fluctuations, weakly expressed | Considerable enhancement of band intensity |
| 721 | No band | Emergence of a band, characterizes pendulum CH-2 fluctuations in long-chain aliphatic structures |
In the test, the stripe 3,300/Am, which confirms the presence of NH groups, was significantly shorter than in control. In the lipid spectra of the calli subjected to radiation, we found considerable weakening of the stripes in the regions between 1,7301,690 cm and 700900/cm proving the presence of heteroaromatic structures. Stripes with the maximum levels of 1,220/cm and 1,050/cm increased considerably in intensity. The presence of these stripes favors the availability of phosphate and ether groups. Of particular interest is emergence of pendulum CH2 fluctuations in the spectra of stripe 721/A< samples subjected to radiation; confirming the presence of long-chain aliphatic structures in transplanar configurations.
On the other hand, the presence of this stripe shows that radiation brought about changes in geometry of lipid matrix, as absorption in this zone means unfolding of heteroaromatic (cyclic) structures into long chains. Therefore,
We conclude that exposure to helium-neon laser light causes remarkable structural changes in membrane lipids. In particular, in IK spectrum of absorption of lipids extracted from irradiated calli registered a stripe of 721/cm, which indicated intensification of the membrane formation processes in the tissues subjected to radiation.
We believe that the reaction of tissue after exposure to laser light has two responses. The first is a primary stress impact, which shows an increase in the number of peroxide oxidation products and a change in membrane-associated enzyme activity. Second are longer secondary reactions connected with adaptive changes in metabolism manifested in structural reconstruction in the membranes, intensification of membrane formation processes, and the stimulation of morphogenetic processes, including regenerative. In both cases, we observed a significant impact of low-intensity laser radiation on membrane structures of plant cells.
References.
N.Yu. Pivovarova, O.I. Grabelnych, T.P. Pobezhimova, N.A. Koroleva, and V.K. Voinikov.
Free fatty acids (FFA) have a number of effects on membranes in general and on mitochondria in particular, where they can significantly affect energy coupling. They increase proton conductance causing dissipation of electrochemical proton gradient. Oxidative phosphorylation uncoupling by free fatty acids in plant mitochondria depends on the function of FFA as protonophores and their interaction with such specific mitochondrial proteins from the family of mitochondrial anion carriers as ADP/ATP-antiporter, plant uncoupling mitochondrial protein (PUMP), and others (Jezek 1999). Fatty acid-dependent uncoupling of oxidative phosphorylation plays an adaptive role during hypothermia and oxidative stress in the plant mitochondria (Casolo et al. 2000; Pastore et al. 2000).
We have shown that such unsaturated (linoleic, oleic, petrozelinic, and erucic) and saturated (lauric, palmitic, stearic, and begenic) fatty acids cause uncoupling of oxidative phosphorylation in winter wheat mitochondria (Grabelnych et al. 2003, 2004, 2005). Using FFA as mitochondrial oxidation substrate was discovered only at early stages of germination of oil-contained seeds such as sunflower and lettuce (Raymond et al. 1992). In our previous work, we found that unsaturated FFA can be used as the sole oxidation substrate for winter wheat mitochondria (Grabelnych et al. 2003, 2004). Data on the influence of FFA on the swelling of animal mitochondria has been published (Schonfeld et al. 2004; Di Paola and Lorusso 2006). At the same time, there is not much data about the influence of fatty acids on plant mitochondria swelling.
This investigation studied the influence of such unsaturated fatty acids as linoleic and linolenic on the winter wheat mitochondria swelling and to determine the role of ADP/ATP-antiporter and plant UCP in this process.
Materials and methods. Three-day-old etiolated shoots of winter wheat (T. aestivum cv. Zalarinka) were germinated on moist paper at 26°C. Mitochondria were extracted from winter wheat shoots by differential centrifugation as describes previously (Pobezhimova et al. 2001). 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, 1 mM MgCl2. Mitochondrial swelling was followed spectrophotometrically by the decrease in optical density (OD) of the mitochondrial suspension (0.25 mg/ml) under deënergized conditions at 26°C at 540 nm. We used the incubation medium as for mitochondrial respiration activity measurement including 125 mM KCl, 18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. Mitochondrial swelling was initiated by FFA addition. FFA concentrations were from 10 mkM to 500 mkM. The concentrations of ADP/ATP-antiporter and uncoupling UCP-like proteins inhibitors were 1 mkM carboxyatractyloside (Catr) and 2 mM GDP, respectively. The concentration of artificial uncoupler carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) was 0.5 mkM. The concentration of mitochondrial protein was analyzed by Lowry method (Lowry et al. 1951). All the experiments were performed on 36 separate mitochondrial preparations. The data were analyzed statistically and arithmetic means and standard deviations are presented.
Results and discussion. Previously, we found that among studied unsaturated fatty acids the most uncoupling activity had C18 FFA, especially linoleic (18:2, n-9, 12) and a-linolenic (18:3, n-3) acids, the addition of which caused a 4- and 7-fold stimulation of nonphosphorylative respiration, respectively. In the present work, we showed that all studied concentrations (10, 50, 100, and 500 mkM) of linoleic and linolenic acids caused effective swelling of winter wheat mitochondria (Figure 1).
Addition of linoleic acid in the incubation medium led to decrease in the optical density (or increase difference between the initial optical density and optical density after given time of incubation) that depended on the fatty acid concentration. Addition of 10 mkM linoleic acid caused an approximately 41% decrease of optical density in 5 min of incubation. The higher concentrations of this acid (50 and 100 mkM) caused more significant decrease of optical density (123% and 129%, respectively) in 5 min of incubation. The maximum decrease in optical density (148%) was observed in experiments with using of 500 mkM linoleic acid and 5 min of incubation. However, the most significant decrease in optical density was observed 20 sec after the addition of linoleic acid. The increase in the optical density difference was 1.5-fold for 10 mkM, 5-fold for 500 mkM, and 6-fold for concentrations 50 and 100 mkM of linoleic acid compared with mitochondria incubated without FFA.
The swelling of winter wheat mitochondria induced by the addition of linolenic acid depended on concentration to a lesser extent (Figure 1). The smallest decrease of optical density in 5 min of incubation was caused by 10 mkM and 50 mkM linolenic acid addition (82% and 89%, respectively). The addition of 100 mkM linolenic acid led to approximately a 101% decrease of optical density in 5 min of incubation. The maximum decrease in optical density in 5 min of incubation was caused by 500 mkM linolenic acid (152%) but with a 10-min incubation, changes in the optical density between control and linolenate-incubated mitochondria decreased. The most significant decrease in optical density was observed in 20 sec after the addition of linolenic acid, similar to experiments with linoleic acid. After 20 sec of incubation of mitochondrial suspension in 10 and 50 mkM linoleic acid, we observed a 3.6-fold and a 3.4-fold increase in the difference of optical densities. Concentrations of 100 and 500 mkM linolenic acid caused approximately 5-fold and 5.4-fold increases, respectively, in the difference of optical densities after 20 sec of incubation of the mitochondrial suspension.
We observed increase of mitochondrial swelling extent in the presence of CCCP that was 2-fold in 20 sec and 5 min incubation (Figure 2). Mitochondria swelling induced by studied FFA were higher than swelling induced by this artificial uncoupler. We suppose that different mechanisms like FFA interaction with ADP/ATP-antiporter and uncoupling UCP-like proteins can participate in FFA-caused winter wheat mitochondria swelling.
To determine the participation of the ADP/ATP-antiporter and plant uncoupling protein in mitochondria swelling induced by C18 fatty acids, we studied the sensitivity of this swelling to inhibitors of the ADP/ATP-antiporter and uncoupling UCP-like proteins. In our experiments, we used FFA concentrations that had the maximum action on winter wheat mitochondria swelling (500 mkM linoleic and 500 mkM linolenic). In experiments with incubating control winter wheat mitochondria with Catr and GDP, we detected a decrease in the optical density of a mitochondrial suspension at 47% and 19%, respectively, after 5 min of incubation (63% and 40% after 10 min of incubation) (Figure 2). Linoleate-induced mitochondria swelling was sensitive to the addition of Catr and GDP. After a 5-min incubation, we observed a 64% and 58% decrease after the addition of Catr and GTP, respectively (Figure 3). Mitochondrial swelling induced by linolenic acid was less sensitive to Catr and GDP addition (Fig. 3). We observed a 49% decrease of linolenate-induced mitochondria swelling by Catr after 5 and 10 min. At the same time, GDP addition inhibited this swelling significantly only after the first 20 sec of incubation (46%). The effect of GDP decreased after 10 min of incubation and was about 14%. Sensitivity of mitochondria swelling induced by linoleic and linolenic acids to inhibitors of the ADP/ATP-antiporter and uncoupling UCP-like proteins indicated their participation in fatty acid-induced swelling.
From the data, we concluded that the uncoupling activity of FFA depends on the chain length and number of double bounds in their molecules. The oxidative phosphorylation uncoupling and mitochondria swelling in presence of FFA are relative processes (Di Paola and Lorusso 2006). Mechanisms of FFA-induced swelling in plant mitochondria deal with the interaction of such specific mitochondrial proteins from the family of mitochondrial anion carriers such as ADP/ATP-antiporter and UCP-like plant proteins.
Acknowledgments. This work was performed in part with the support of President Russian Federation grant for state support of leading scientific schools of the Russian Federation (SS-4812.2006.4), an interdisciplinary integration project of the Russian Academy of Sciences N47, the Russian Science Support Foundation, and the Siberian Division of Russian Academy of Sciences Youth Grant (project 115).
References.
N.S. Pavlovskaya, O.V. Savinova, O.I. Grabelnych, T.P. Pobezhimova, N.A. Koroleva, and V.K. Voinikov.
The uncoupling of oxidative phosphorylation and swelling are events that precede the opening of the
permeability transition pore (PTP) in mitochondria, the release of cytochrome c, and the induction of programmed cell
death (Crompton 1999; He and Lemasters 2002; Tsujimoto et al. 2006). Recent evidence suggests that mitochondrial
volume seems to affect mitochondrial electron transport, reactive oxygen species
production, cytochrome c release in the
process of apoptosis, and participates in mechanical signaling pathways (Kaasik et al. 2007). Lim et al. (2002) showed that
an increase in the matrix volume correlates well with an increase in respiration rate. The induction mechanism of PTP
is poorly understood. In plants, PTP is both sensitive to CsA (Arpagaus et al. 2002; Tiwari et al. 2002) and
insensitive (Fortes et al. 2001; Curtis and Wolpert 2002; Virolainen et al. 2002).
Data about influence of stress factors for opening of PTP in plant mitochondria are lacking.
Our previous study showed that CsA causes a decrease in state-4 respiration in winter wheat mitochondria, and its influence is substrate-specific (Grabelnych et al. 2004). We detected the most pronounced effect of this treatment in mitochondria in the presence of Ca2+ ions. Furthermore, we detected the stimulation of swelling in winter wheat mitochondria from nonstressed seedlings shoots by Ca2+ ions and palmitic acid and the inhibitory effect of CsA (Pavlovskaya et al. 2006). Pavlovskaya et al. (2006) found that CsA inhibits the Ca2+-induced swelling of mitochondria from nonstressed shoots but did not inhibit the Ca2+-induced swelling of mitochondria from cold-stressed and cold-hardened shoots. The data allowed us to suggest the existence of a CsA-insensitive, mitochondrial pore function in winter wheat shoots in conditions of cold stress and hardening. The involvement of the mitochondrial cyclosporin A-insensitive pore induced by palmitic acid and Ca2+ ions complexes in the apoptotic process of animal cells was shown (Belosludtsev et al. 2006).
Our aim was to study respiration and swelling in the presence of inductors (Ca2+ ions and palmitic acid) and an inhibitor (cyclosporin A) of PTP in mitochondria from cold-stressed and cold-hardened shoots of cold-resistant winter wheat after subsequent oxidative stress.
Material and methods. Three-day-old etiolated seedlings of cold-resistant winter wheat
(T. aestivum subsp. aestivum cv. Zalarinka) were germinated on moist paper at 26°C. Seedlings were subjected to short-term (-4°C, 1 h) cold
stress with subsequent oxidative stress or cold hardening for 7 days at 4°C with subsequent oxidative stress. Oxidative
stress was induced by immersing root tips of intact 3-day-old etiolated seedlings in 0.5 mM solution of
H2O2 in the dark at 26°C for 4 h. The mitochondria were isolated from seedling shoots by differential centrifugation (Pobezhimova et
al. 2001), and their energetic activity and swelling were studied. The isolated mitochondria were resuspended in
the following medium: 40 mM MOPSKOH buffer (pH 7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1
mM MgCl2. Mitochondria activity was recorded polarographically at 26°C using a closed-type platinum electrode in a 1.4
ml cell volume. The reaction mixture contained 125 mM KCl, 18 mM
KH2PO4 (pH 7.4), 5 mM EDTA, and 1 mM
MgCl2. Oxidation substrates were 10 mM malate in the presence of 10 mM glutamate, 8 mM succinate in the presence of 5
mM glutamate, and 1 mM NADH. During succinate and NADH oxidation, 3 mkM rotenone was added to the
incubation medium. Polarograms were used to calculate the rates of phosphorylative respiration (state 3),
nonphosphorylative respiration (state 4), respiratory control
by Chance-Williams, and the ADP:O ratio (Estabrook 1967). Mitochondrial swelling was
followed spectrophotometrically by the decrease in
optical density (OD) of the mitochondrial
suspension (0.25 mg/ml) under deënergized conditions
at 26°C at 540 nm. We used an incubation
medium containing 200 mM KCl and 20 mM MOPS (pH 7.4). The following concentrations of
test reagents were used: 1 mkM cyclosporin A,
1.75 mM Ca2+, and 50 mkM palmitic acid. In
the experiments using CsA and Ca2+, the
preincubation time was 5 min at 0°C. The concentration
of mitochondrial protein was analyzed according to Lowry et al. (1951). Results are represented
as the mean of at least three
determinations/experiment.
Results and discussion. The study of
respiration in the mitochondria isolated from control
(nonstressed), cold-stressed with subsequent
oxidative stress, and cold-hardened with
subsequent oxidative stress in winter wheat shoots
showed that it was sensitive to CsA (Figure 4). Ions of
Ca2+ stimulated nonphosphorylative respiration (state
4) in mitochondria except for mitochondria of cold-stressed with subsequent oxidative
stress seedlings (Figure 4). The Ca2+-induced increase
in respiration rate was CsA sensitive (Figure 4).
The addition of palmitic acid did not cause a
significant increase of respiration in mitochondria
both cold-stressed with subsequent oxidative stress
and cold-hardened with subsequent oxidative one winter wheat
shoots (Figures 4b and 4c). Palmitate-induced respiration of the control
mitochondria was CsA sensitive (Figure 4). The results
also indicated substrate dependence with respect
to action of inhibitor and inductors of the
mitochondrial pore. Mitochondria of the control
seedling shoots were the most sensitive to the addition of CsA,
Ca2+ ions, and palmitic acid during
oxidation of malate (Figure 4a). At the same time, sensitivity to the addition of these reagents
in mitochondria isolated from cold-stressed with
subsequent oxidative stress and cold-hardened with subsequent oxidative stress in winter wheat
shoots was the most during succinate or NADH
oxidation (Figures 4b and 4c).
In experiments with incubated mitochondria isolated from control winter wheat shoots with CsA, we detected the decrease in the optical density of the mitochondrial suspension after 5 min of incubation (34%, Figure 5). The influence of Ca2+ ions on the swelling of winter wheat mitochondria was studied on mitochondria that were preliminarily incubated with and without CsA. The presence of Ca2+ in the incubation medium stimulated the extent of swelling (3 fold) in control winter wheat mitochondria compared to swelling of mitochondria incubated without Ca2+ (Figure 5). The Ca2+-induced swelling was fully inhibited after a preliminarily incubating mitochondria with CsA (Figure 5). We observed an increase in the swelling extent of the control winter wheat mitochondria in the presence of palmitic acid, the effect of that was similarly to Ca2+ action. Palmitic acid caused a 4-fold increase of swelling after 5 min of incubation, which was sensitive to CsA addition (Figure 5).
Cold stress with subsequent oxidative stress and cold hardening with subsequent oxidative stress of winter wheat seedling shoots increased the extent of mitochondrial swelling compared with control (Figure 5). This swelling is fully inhibited by CsA. Ions of Ca2+ and palmitic acid did not influence on swelling of mitochondria isolated from cold-stressed plants with subsequent oxidative stress and cold-hardened plants with subsequent oxidative stress (Figure 5). For mitochondria isolated from cold-hardened winter wheat shoots with subsequent oxidative stress, the ions of Ca2+ even inhibited the swelling (Figure 5).
Based on our previous work, short-term cold stress and cold hardening decrease the sensitivity of mitochondrial swelling to CsA both in the absence and presence of Ca2+ ions (Pavlovskaya et al. 2006) and the present work regarding oxidative stress followed by short-term cold stress and cold hardening causes the appearance of mitochondria sensitive to the action that the mitochondrial pore inhibitor, we can propose a function of the CsA-sensitive mitochondrial pore in winter wheat shoots in normal conditions and under oxidative stress. Different mechanisms seem to be responsible for the PTP function. Accordingly, studying the influence of a single oxidative stress on mitochondria function in winter wheat shoots and its effect on the sensitivity of respiration and swelling to inductors and inhibitors of mitochondrial pore is necessary.
Acknowledgments. This work was performed, in part, with the support of President Russian Federation grant for state support of leading scientific schools of Russian Federation (SS-4812.2006.4), interdisciplinary integration project of Russian Academy of Sciences No. 47, the Russian Science Support Foundation, the Russian Foundation of Basic Research (project 05-04-97231), and the Siberian Division of Russian Academy of Sciences Youth Grant (project 115).
References.
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.
T.I. Odintsova, V.A. Pukhalskiy, T.V. Korostyleva, and G.V. Kozlovskaya (Vavilov Institute of General Genetics) and A.K. Musolyamov and Ts.A. Egorov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry).
All living organisms have evolved mechanisms to defend themselves against pathogen attack. Although plants do not have an immune system, they synthesize a variety of defense molecules including low-molecular-weight compounds, proteins, and peptides with antifungal and antimicrobial activity. These proteins and peptides are involved in either constitutive or induced resistance to fungal or bacterial attack. Hundreds of antifungal/antibacterial peptides and proteins are known and, among them, antimicrobial peptides play an important role.
Defensins are small basic cysteine-rich peptides (4547 amino acid residues long) that form an amphiphilic structure (Thomma et al. 2002). They play diverse roles in nature, showing antibacterial and/or antifungal activity (Lay and Anderson, 2005) and the capability to inhibit insect a-amylases and proteinases (Melo et al. 2002). Defensins are encoded by multigene families. In recent studies, more than 300 defensin-like genes have been identified in the Arabidopsis genome (Silverstein et al. 2005). Defensins are attractive candidates for the control of pathogenic microorganisms and pests by genetic engineering of crops.
In our previous studies, we showed that Triticum kiharae, a synthetic allopolyploid produced by crossing T. timopheevii subsp. timopheevii with Ae. tauschii contains a variety of antimicrobial peptides, 24 of which were new (Egorov et al. 2005). We showed that defensins of this species comprised at least 13 components that differed in their N-terminal sequences. Three subgroups of defensins were discriminated. In this work, we completely sequenced the so-called D defensins, which belong to a new previously unknown group, and isolated defensins in seeds of the presumable of A-, B-, and D-genome donors to polyploid wheat.
Material and methods.Seeds of several species were used in this study: Triticum kiharae Dorof. et Migush., T. monococcum subsp. monococcum (AA), Ae. speltoides (BB), and Ae. tauschii (DD). Wheat flour was defatted with petroleum ether (1:10) and extracted with an acid solution (1 M HCl and 5% HCOOH) for 1 h at room temperature and desalted on a Aquapore RP300 column. Freeze-dried, acidic extract was subjected to chromatography on Heparin Sepharose. Proteins and peptides were eluted with a stepwise NaCl gradient. The 100-mM NaCl fraction was collected, desalted as described above and separated on a Superdex Peptide HR 10/30 column (Amersham, Pharmacia, Biotech, Uppsala, Sweden). Proteins and peptides were eluted with 0.05% TFA, containing 5% acetonitrile at a flow rate of 250 l/min and monitored by absorbance at 214 nm. The peptide fraction was further separated by RP-HPLC on a Reprosil C18 column (4.6 x 250 mm, particle size 5 m) with a linear acetonitrile gradient (1050%) for 1 h at a flow rate of 1 ml/min and 40°C. Peptides were detected at 214 nm. Mass spectra were acquired on a MALDI-TOF mass spectrometer (Micromass, UK). Amino acid sequencing was performed by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems).
Results and discussion.From seeds of T. kiharae and related species, eight new defensins were isolated from the 100-mM fraction and completely sequenced (Table 1).
Peptide |
Amino acid sequence | Molecular mass (Da) |
|---|---|---|
 |
||
Sequence alignment of wheat D defensins with those of other cereals with a CLUSTAL W program showed the highest homology with TAD1, a defensin specifically induced in wheat during cold acclimation (Koeke et al. 2002). D defensins share sequence similarity with defensins from other Poaceae species, but differ considerably from those of other plant families. Analysis of defensins from diploid species, the putative donors of A, B, and D genomes to polyploid wheat, showed that all of them possess D defensins. The number of defensins identified in each diploid species and expressed in seeds suggests the existence of at least three defensin-encoding genes in each genome. Sequence data and mass analysis showed that the structure of D defensins is highly conserved during at least 10,000 years of separate evolution of diploid and polyploid forms. Most defensins are genome-specific, namely present in a single genome type (A, B, or D genome), allowing us to locate most D-defensin-encoding genes: D1-group defensins, as well as D4 and D5, are A-genome encoded; D3 and D6 are D-genome encoded; and D6.1 and D3.1 (D3 homologue) are B-genome encoded. The origin of D2 defensin remains unclear, because it was found both in B and D genome. We speculate that this defensin type emerged in the evolution before the divergence of Aegilops species and was preserved in hexaploid forms.
Acknowledgment. This work was supported in part by RFBR grants no. 06-04-48874 and no. 05-04-49565 and the Program of the Russian Academy of Sciences 'Gene pool dynamics in plants, animals and humans'.
Reference.
E.N. Bilinskaya, S.P. Martinov, A. Dragovich, S. Dencic, and V.A.Pukhalskiy.
Introduction. The distribution of hybrid necrosis genes in winter wheat cultivars of the former Yugoslavia was first published in the works of Hermsen (1963) and Tsunewaki and Nakai (1976). All the genotypes studied were noncarriers of hybrid necrosis genes (ne1ne1ne2ne2 genotype). In subsequent studies, the data on necrotic genotypes of the recognized cultivars of the former Yugoslavia and of the selection lines produced in different years appeared (Zeven 1969, 1971, 1973, 1976, 1981; Shoran et al. 1983; Dimitrijevic 1988; Kochumadhavan et al. 1988; Jost et al. 1989). Following Hermsen (1963) and Tsunewaki and Nakai (1976), the necrotic genotypes of 53 cultivars and lines of winter wheat were described. Thirty-eight cultivars (71.1%) had the ne1ne1ne2ne2 genotype, nine cultivars (17.0%) possessed the Ne1Ne1ne2ne2 genotype, and six cultivars (11.3%) had the ne1ne1Ne2Ne2 genotype. The cultivars studied were developed in different years beginning from 1919 (the cultivar Non plus ultra) to the beginning of the 1980s (the cultivars Sava, Zitnica, and Sutjeska). The studies of changes in necrotic genotype frequencies in modern populations of this region caused by breeding strategy are of particular interest.
Materials and methods. We studied the distribution of necrotic genotypes in 32 cultivars of winter wheat produced in two breeding centers in Serbia, the Institute of Cereal and Vegetable Crops (Novi Sad) and the Cereal Center (Kragujevac). Winter wheat cultivars Co 725872 (Ne1Ne1ne2ne2 genotype) and Mironovskaya 808 (ne1ne1Ne2Ne2 genotype) were used as testers. Crossing was in the field by conventional procedures including emasculating and isolating spikes. Plants of the first hybrid generation were examined for the symptoms of hybrid necrosis. The strength of hybrid necrosis alleles was evaluated according to Hermsen (1963).
Results and discussion. Of the 31 cultivars studied (Table 1), five had the genotype ne1ne1Ne2Ne2 (Licanka (Ne2w), Senica (Ne2w), Selecta (Ne2w), Ibarka (Ne2w), and Orasanka (Ne2m). The first four cultivars were bred in Novi Sad and have a weak allele w of the Ne2 gene, whereas Oransanka is from the Cereal Center and has the moderate allele m. This difference is most likely due to the specific original selection material used in these breeding centers. The data shown in Figure 1 demonstrate different approach to the selection of the original material. In both centers, noncarriers of hybrid necrosis genes were selected (ne1ne1ne2ne2 genotype). In all probability, such forms have selective advantages over the carriers of hybrid necrosis genes in this region, which follows from the fact that during selection the presence or absence of hybrid necrosis genes was not taken into account. The Ne1Ne1ne2ne2 genotype was not found among the cultivars studied.
Acknowledgment. This work was supported in part by the Program of the Russian Academy of Sciences 'Gene pool dynamics'.
References.
V.A. Pukhalskiy, E.N. Bilinskaya, S.P. Martynov, and L.A. Obolenkova.
Introduction. Modern breeding contributes considerably to the distribution of hybrid necrosis genes in wheat populations in different countries (Altukhov et al. 2005). The genetic erosion is clearly seen that dictates the necessity to develop a new strategy of breeding and formation of gene bank collections. Therefore, the study the microevolution of the wheat genome using different genetic markers including hybrid necrosis genes is interesting. Here, we present data on the analysis of necrotic genotypes in the former Czechoslovakia and modern Czech Republic.
Materials and methods. Thirty cultivars of winter wheat released in different breeding centers of the former Czechoslovakia and the Czech Republic were studied. Mironovskaya 808 (ne1ne1Ne2Ne2 genotype) and Co725082 (Ne1Ne1ne2ne2 genotype) were used as testers. Crosses were made in the field by conventional procedures with isolation of spikes. F1 hybrids were grown in the field, and the symptoms of hybrid necrosis were evaluated at different ontogeny stages.
Results and discussion. Our results on the presence of hybrid necrosis genes in wheat cultivars are given in Table 2. In the cultivars studied, the Ne1 gene was absent, whereas 66.7% of the cultivars had the ne1ne1Ne2Ne2 genotype. The ne1n51n52ne2 genotype was identified in 33.3% of the wheats. Because we investigated wheat cultivars produced between 1990 and 2003, we compared our data with the results obtained earlier (Apltauerova 1983; Zeven 1969, 1973, 1976, 1981; Sarkisyan 1972; Sarkisyan and Petrosyan 1972; Pukhalskiy et al. 1997). In the literature, the necrotic genotypes of 30 cultivars of winter wheat of the former Czechoslovakia have been described. Two of these cutivars (6.7%) have Ne1, whereas 14 (46.7%) were Ne2-carriers, and 14 (46.7%) were noncarriers of hybrid necrosis genes. The Ne1 gene was identified in the cultivar Iva released in 1962 and in the selection line ST-46-78 (Apltauerova 1983).
Cultivars produced in the former Czechoslovakia between 1915 and 1930 were noncarriers of hybrid necrosis genes and included Diosecka (1915), Chemecka 12 (1919), Dobrovicka (?), Valtcka osinata (1936), and Diosecka Nova (1930) (Zeven 1981). Hybrid necrosis genes probably appeared in the breeding centers of the former Czechoslovakia after the 1950s.
Our data, together with the findings of other researchers, indicate a dramatic increase in the ne1ne1Ne2Ne2 genotype frequencies among winter common wheat cultivars of the former Chech Republic. Similar processes occur in other wheat-growing regions of the world (Altukhov et al. 2005; Pukhalskiy and Bilinskaya, 2006). This tendency is still difficult to explain. Another interesting peculiarity is the high frequency of strong alleles s (45%) and ms (20%), although the frequency of the m allele was 15%, of the mw allele 10%, and of the w allele 10%. This is a rare case of a relatively small population, especially if we take into consideration that according to Zeven (1976), the former Czechoslovakia belonged to a region where noncarriers of hybrid necrosis genes were predominantly cultivated (ne1ne1 ne2ne2 genotype). Cluster analysis of the relatedness coefficients showed rather low variation among the cultivars with the strong allele of the Ne2 gene (Figure 2). In 9 out of 10 instances, the Mironovskaya 808 (Ne2ms) gene was the donor of strong alleles. This cultivar is in the pedigree of Vega, Ina, Boka, Brea, Bruneta, Saskia, Banquet, Senta, and Sofia. Together with Mironovskaya 808, this allele could have been from Noe via Maris Huntsman (Sofia) and Turkey via Benno (Senta). Mironovskaya 808 is absent only from the pedigree of Meritto. In this case, the donor of the Ne2s allele could be Noe through Maris Huntsman and Heines VII or Turkey through Carstens VIII. We cannot exclude that in this case, together with certain alleles of the Ne2 gene, some other genes located on the D genome that function as catalyzers or minor promoters were involved in breeding (Jha et al. 1980).
Acknowledgment. This work was supported in part by the Program of the Russian Academy of Sciences "Gene pool dynamics".
References.