A Database for Triticeae and Avena
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.
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.
Table 1. The energetic activity of winter
wheat mitochondria isolated from control (1), stressed (2), and
hardened (3) shoots analyzed using different oxidizing substrates.
Data are presented as mean + standard error, n = 632.
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
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).
Table 2. The contribution of cytochrome pathway
(Cyt) or alternative pathways with cyanide-resistant alternative
oxidase (Alt(AOX)), CSP 310 (Alt(CSP310)), and outer NADH-dehydrogenase
(NADH(outer)) to total respiration of winter wheat mitochondria
in control conditions (1), during short-term cold stress (2),
and during hardening (3) using different oxidizing substrates.
The contribution is expressed as a percent of the respiratory
rate in state 4.
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).
O.I. Grabelnych, T.P. Pobezhimova, A.V. Kolesnichenko, and
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
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).
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
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
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
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
Table 3. Substrate specificity of the enzymes
of amino acid metabolism, % to conversion of tryptophan (Try).
Experiments were repeated at least twice. Substrate specificity
of the enzymes of amino acid metabolism, % to conversion of tryptophan
(Try). Experiments were repeated at least twice.
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 specific activity of UDPG-transferase
in wheat shoots, nmol of IAA glucosyl ester/mg of protein/h.
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
G.B. Borovskii, A.Yu. Yakovlev, S.V. Vladimirova, and V.K.
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
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,
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
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.
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
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
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
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
Table 6. Wet weight of roots and of the aerial
portion, and mean length of 15 spring wheat seed lings as a function
of soil temperature and N-form. Air temperature was the same for
all variants, 19 ± 1 C.
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
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 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.
Table 1. Molecular mass of the RP-HPLC fractions
obtained from the fungicidal fraction D. Prevailing masses are
indicated in bold.
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
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
These results indicate that T. kiharae possesses different
types of antimicrobial peptides.
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
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.
Table 2. Genotype of necrosis genes identified
in modern Australian cultivars of common spring wheat.
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
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
N.I. VAVILOV RESEARCH INSTITUTE
OF PLANT INDUSTRY
42, B. Morskaya Str., St. Petersburg, 190000, Russian Federation.
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.
Table 3. Analysis of variance of the contribution
of hypothetical sources of resistance to common bunt for Russian
and Ukrainian winter wheat cultivars. Factor A is the group of
resistant cultivars, factor B is the geographical region of origin,
and factor C is the ancestry. * = significance at P < 0.0001.
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.
Table 4. Average contribution of hypothetical
sources of resistance to common bunt for Russian and Ukrainian
winter wheat cultivars in groups of resistant and susceptible
accessions. Values are followed by letters that indicate significant
differences at P < 0.05 by Duncan's multiple range test.
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.