The effect of wheat chromosome 5A on free amino acid accumulation induced by osmotic stress was compared in chromosome 5A substitution lines with different freezing tolerance. Treatment with 15% polyethylene glycol (PEG) resulted in greater total free amino acid content even after 3 days compared to the controls. The ratio of amino acids belonging to various amino acid families differed after 3-week treatment in the control and PEG-treated plants only in the case of the freezing-sensitive substitution line. There was a transient increase with a maximum after 3 days in the amounts of several amino acids, after which their concentrations exhibited a more gradual increase. During the first days of osmotic stress the Glu, Gln, Asp, Asn, Thr, Ser, Leu, and His concentrations were greater in the tolerant substitution line than in the sensitive one, while the opposite relationship was observed at the end of the PEG treatment. The coordinated changes in the levels of individual amino acids indicated that they are involved in both the short- and long-term responses to osmotic stress. The alterations differed in the two chromosome 5A substitution lines, depending on the stress tolerance of the chromosome donor genotype.
Abiotic stresses result in severe injury and yield loss in crops. A common consequence of drought and low temperature is the induction of osmotic stress. The accumulation of compatible solutes, such as certain amino acids especially Pro, prevents the development of severe osmotic stress. Amino acids also have several other roles in plants, for example they regulate ion transport and stomatal opening and affect the synthesis and activity of enzymes, gene expression, and redox homeostasis, helping the plants to cope with the harmful effects of osmotic stress [
Osmotic stress, which can be induced by limited water supply or osmotic agents, alters both the amino acid pattern and the concentrations of individual amino acids. Osmotic stress induced by polyethylene glycol resulted in an increase in the concentrations of Asp, Glu, Asn, Thr, Ser, Ala, and Pro during the first day of treatment in the roots of maize [
Proline plays a very important role as an osmoprotectant in the adaptation to osmotic stress [
Besides Pro, the involvement of other amino acids in the response to osmotic stress was also demonstrated. Arginine was found to function as a compatible solute, improving stress tolerance in yeast [
Chromosome 5A of wheat was found to affect the cold-induced accumulation of certain free amino acids [
A specific genetic system consisting of the moderately freezing-sensitive wheat (
Seedlings were grown in hydroponic culture using half-strength modified Hoagland nutrient solution [
Shoot samples of 300–600 mg fresh weight were crushed in liquid nitrogen and extracted with 2 mL cold 10% trichloroacetic acid for 1 hour with gentle agitation on a shaker (C. Gerhardt GmbH & Co. KG, Germany) at room temperature. Each sample was filtered through a 0.2
The free amino acid data were evaluated using analysis of variance and the significant differences were calculated.
Application of PEG in 15% concentration resulted only in mild osmotic stress as it was shown by growth data (Figure
Effect of osmotic stress on fresh ((a); significant difference (SD): 0.612) and dry weight ((b); SD: 0.065). The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency).
The total free amino acid concentration increased already after 3 days osmotic stress in all the genotypes, but a great difference between control and treated plants was observed after 3 weeks, especially in the case of CS and CS(Tsp5A) (Figure
Effect of osmotic stress on total free amino acid content. The time course of changes was compared under control ((a); SD: 4.47) and stress (15% PEG; (b); SD: 12.68) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes.
Changes in the ratio of amino acid families during osmotic stress. A comparison was made of the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes, grown for three weeks under control (C) or stress conditions (15% PEG). The experiment was repeated with three parallels three times with similar results.
In the case of the individual amino acids little or no change was observed during the 3 weeks of treatment in the control plants, while the level of most of the 19 amino acids examined was increased by osmotic stress (Figures
Effect of osmotic stress on the concentration of amino acids belonging to the glutamate family. The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency). (a): Glu (SD: 1.66), (b): Gln (SD: 5.10), (c): Pro (SD: 1.67), (d): Arg (SD: 0.070), and (e):
Effect of osmotic stress on the concentration of amino acids belonging to the aspartate family. The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency). (a): Asp (SD: 0.505), (b): Asn (SD: 5.92), (c): Lys (SD: 0.037), (d): Ile (SD: 0.050), and (e): Thr (SD: 0.204).
Effect of osmotic stress on the concentration of amino acids belonging to the serine family. The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency). (a): Ser (SD: 0.644), and (b): Gly (SD: 0.159).
Effect of osmotic stress on the concentration of amino acids belonging to the pyruvate family. The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency). (a): Ala (SD: 0.261), (b): Val (SD: 0.146), and (c): Leu (SD: 0.058).
Effect of osmotic stress on the concentration of amino acids belonging to the aromatic amino acid family. The time course of changes was compared under control and stress (15% PEG) conditions in the freezing-sensitive CS and CS(Tsp5A) and the freezing-tolerant CS(Ch5A) wheat genotypes. The standard deviations of the data were below 10% in each case (not shown on the graphs because of the better transparency). (a): Tyr (SD: 0.046), (b): Phe (SD: 0.091), (c): His (SD: 0.122), and (d): 3-metil-His (SD: 0.026).
The glutamate content exhibited a very rapid PEG-induced increase during the first three days of treatment, but later its concentration transiently decreased, then increased again in CS and CS(Ch5A) (Figure
The aspartate concentration exhibited a transient increase during the first days of the experiment with a local maximum after 3 d of treatment in CS(Ch5A) and CS (Figure
Two members of serine amino acid family could be detected. The Ser concentration exhibited a large transient increase during the first week of the treatment in CS and CS(Ch5A), while it gradually increased in CS(Tsp5A) during the 3-week osmotic stress (Figure
All 3 members of the pyruvate family were studied. The Ala concentration showed greater changes only in CS, where it had a transient increase during the first week, and increased again during the second half of the experiment (Figure
Four aromatic amino acids could be measured. The Tyr concentration, after an initial transient increase, exhibited a rapid increase in CS(Tsp5A) and a slow one in the other two genotypes (Figure
Interestingly, the fresh and dry weight data of control and treated plants did not differ at the end of the 3-week treatment with 15% PEG in the present experiment, however on the basis of shoot damage monitored after two weeks recovery phase following the 3-week osmotic stress in a previous study, the 3 genotypes had different level of tolerance [
Despite the great changes induced by osmotic stress in the amounts of most amino acids, the ratio of the amino acid families was similar after 3 weeks in the control and PEG-treated plants except for CS(Tsp5A), indicating that the alterations observed were coordinated. A similar coordination was found in the level of minor amino acids in the leaves of wheat and potato grown under various photosynthetic conditions [
In the case of many amino acids (Glu, Gln, Asp, Thr, Ser, Gly, Ala, Tyr) a rapid transient increase was first observed with a local maximum after 3 days of PEG treatment, after which their levels increased again from the 7th day of osmotic stress. These results suggest that an elevation in the amino acid content is involved in both the short- and long-term response to osmotic stress. The initial increase was 1.5-2-fold greater in the case of several amino acids in the freezing-tolerant CS(Ch5A) than in the freezing-sensitive CS(Tsp5A) after osmotic stress (present study) and cold [
The special role of Pro in the stress response, described in several publications [
In contrast to several other amino acids, there was no further great increase in the Glu content after the initial transient rise. This can be explained by its intensive use for the synthesis of Pro, polyamines, and GSH during the long-term (3-week) stress response. This assumption is confirmed by the observation that the spermine content increased in wheat after 3 weeks of osmotic stress [
The protective role of Arg during osmotic stress, indicated by the fact that its level changed to different extents in wheat genotypes with different tolerance, may be due to its osmoprotective function, observed in yeast [
The comparison of two chromosome 5A substitution lines with different levels of stress tolerance revealed that this chromosome affects both the short- and long-term changes in the free amino acid concentration. This effect is probably based on the regulatory role of the Cbf/Dreb (C-repeat binding/drought-responsive element binding) transcription factors mapped at the
In summary, coordinated changes in the levels of individual amino acids indicate that they are involved in both the short- and long-term response to osmotic stress. The alterations differed for the two chromosomes 5A substitution lines, depending on the stress tolerance of the chromosome donor genotype.
The authors wish to thank A. Horváth and N. Csabai for their help in plant cultivation and treatment and K. Hetes-Lőrincz for technical work in the analysis of free amino acids. This work was supported by the European Union (AGRISAFE 203288-EU-FP7-REGPOT2007-1), by the Hungarian Scientific Research Fund (OTKA K83642), and by the Hungarian National Development Agency (TÁMOP-4.2.2.B-10/1-2010-0025).