Action of surfactants on the biochemical and morphometric parameters of soft wheat (Triticum aestivum L.)

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Action of surfactants on the biochemical and morphometric parameters of soft wheat (Triticum aestivum L.)

Omirbekova N.Zh., Zhussupova A.I., Shulembayeva K.K.

al-Farabi Kazakh National University, Almaty,

e-mail: omirbekova.nargul@kaznu.kz

Abstract

proline protein wheat mutant

The effect of 1% aqueous solutions of various surfactants on content of free proline and protein, activity of some enzymes of nitrogen and energy metabolism and quantitative changes in anatomical structure of wheat have been studied. Significant accumulation of free proline was shown in roots of wheat plants of Shagala variety exposed to surfactants. It was found out that protein content in seeds of a number of mutant lines increased from 5 to 9%, in compare with the initial varieties Shagala and Zhenis.

Mutants, induced by surfactants, had a significantly higher or lower enzymatic activity in comparison with the original varieties. Mutant lines 1, 7, 23 and 24 represented breeding value on the basis of "leaf pubescence".

Introduction

Kazakhstan is one of the largest grain-producing countries. Area, occupied by major food crop - wheat, is more than 14 million hectares. One of the main factors that increase the grain harvest is a way of breeding - creating high-yielding varieties with the complex of valuable economically important traits and properties.

Success in solving this problem depends on the level of genetic diversity of initial collections of hybrid populations, as well as on methodology of selection of desired genotypes within the breeding process.

Directed mutational variability can be induced by chemical compounds that are involved in plant metabolism and have regulatory mode of action [1]. Chemical compounds, that cause variability with a positive effect, should be picked as mutagens. One of possible choices is surfactants.

Results of experiments on various biological effects and violations of the structure and functions of organisms exposed to synthetic surfactants can be found in literature [2, 3]. Thus, the effect of different concentrations of aqueous solutions of sodium dodecyl sulfate on the viability of aquatic plants (Potamogeton crispus L.) showed that the surfactant influenced morphological characteristics of plants, depending on the level of exposure [3].

Study of wheat growth under the influence of anionic detergent and nonionic surfactant showed both positive and negative effects, depending on concentration and time of treatment. The reason for stimulation of growth is still not clear. However, the authors have shown that surfactants can have different effects on the organism: interact with structural proteins and enzymes, with cytomembranes, improve absorption of auxin, dissociate the chlorophyll-protein complex, and suppress the synthesis of proteins and DNA [4].

The study of genetic and biochemical characteristics of induced mutants provides an opportunity not only to clarify the mechanisms of mutational variability, but also resolve issues related to the rational use of induced mutants in plant breeding [1].

Plants with altered quantitative and qualitative characteristics, steadily inherited through generations M₁-M₃, have been previously obtained using preliminary treatment of seeds with water solutions of surfactants. For wheat varieties Zhenis and Shagala plants of M₁ generation were obtained, characterized by different morphological characteristics: forms of spikes, grains, length of stem, productive tillering, increased number of grains and mass of the main spike, awnless or awned, anthocyanin color, etc.

The aim of the current study was to analyze the effect of surfactants on the contents of free proline in wheat seedlings, activity of some enzymes of nitrogen and energy metabolism of the mutant lines and on anatomical structure to obtain reliable information on the value of obtained genotypes.

Materials and methods

The objects of the study were seedlings grown for 14 days from seeds, treated with surfactants. Seeds of common wheat varieties Zhenis and Shagala served as a control.

To obtain 14 day-old seedlings, seeds were previously soaked in 1% aqueous solutions of surfactants for 5 hours under the temperature of 25 ⁰C. Treated seeds were washed for 30 minutes under tap water, then three times for 5 min with sterile water. Seeds were germinated for 48 hours in Petri dishes on moistened filter paper in an incubator under the temperature of 25 ⁰C, and then at room temperature under the light.

Before sowing seeds were treated with 1% aqueous solutions of surfactants commonly used in biological research: Triton X-305, Triton X-100, Tween 85, Tween 65, and Tween 20. Exposition has taken 5 hours under the temperature of 25 ⁰C. After the treatment, seeds were washed for 30 minutes under the tap water and dried. Planting has taken place in the fields of KazSRI of farming and crop production. Untreated dry seeds of the initial variety served as a control.

Content of free proline in the vegetative organs of seedlings was calculated according to the method of L. Bates et al. [5]. To determine the content of proline the calibration curve ranging from 0.01 to 0.2 mM was constructed (Ajinomoto, Japan). The total protein content was found using microbiurete method by Bailey at a wavelength of 330 nm [6].

Enzymatic activity was analyzed on the basis of standard methods [7]. Changes in enzymatic activity were detected by changes in the levels of absorption at 340 nm on a spectrophotometer Ultrospec 1100 pro (Biosciences Amersham, UK).

Reaction mixtures for defining the activity of malate dehydrogenase (MDG) contained NAD and malate, for alcohol dehydrogenase (ADH) - NAD, and 96% ethanol, for glutamate dehydrogenase (GDH) - NADH, 2-oxoglutarate, ammonium sulfate, for the enzymatic complex of malate dehydrogenase and glutamate oxaloacetate aminotransferase (EC MDG -GOAT) - malate, NAD, glutamate.

For anatomic studies, vegetative plant organs were fixed by standard procedures [8]. Microphotography of objects of study was performed using microscopes (Axioskop 40, Carl Zeiss) and digital camera Canon Power Shot S45.

Statistical processing has been done according to Rokitsky P.F. [9].

Results and discussion

One of the adaptive features of plants to stress factors is the accumulation of proline in the cells. Accumulation of proline may be caused by activation of synthesis of this amino acid, inhibition of its degradation or enhancing the hydrolysis of proteins containing large amounts of proline [10]. The speed of the mechanisms of adaptation to stress factors is in tight connection with the ability of plants to induce rapid accumulation of proline in response to stress [11].

Given the important role of proline in the process of adaptation to abiotic factors, its level in wheat seedlings, treated with 1% aqueous Triton X-100 solution, was studied (Table 1).

Table 1 - Effect of 1% aqueous solution of Triton X-100 surfactant on the level of free proline accumulation in the vegetative organs of Shagala variety seedlings.

As shown in the Table 1, content of free proline in leaves and stems of wheat seedlings slightly decreased, in compare to control. Significant accumulation of free proline in the roots of affected Shagala variety wheat plants was shown. Thus, the accumulation of proline in roots (1.95 mg/g) increased by more than 3 times in compare with control (0.60 mg/g).

High accumulation of free proline in the roots indicated protective reaction of wheat to the action of Triton X-100 surfactant. The results obtained confirm the literature data that the high content of free proline in seedlings is an indicator of resistance to abiotic factors [10].

Comparative study on the content of soluble protein in seeds of mutant forms and the original wheat varieties Shagala and Zhenis has been performed.

It was shown that in mutant line 3 (dense short swords), mutant line 4 (large grain and the absence of spines), and mutant line 5 (long ear), content of protein increased from 5 to 9 % in compare with its content in original Shagala variety plants.

The content of soluble protein in the seeds of mutant line 23 (elongated grains, awned form) was by 9% higher (0.210 mg/ml), than that of the initial variety Zhenis - 0.192 mg/ml (Table 2).

Table 2 - Activity of enzymes of nitrogen and energy metabolism in mutant lines and original wheat varieties Shagala and Zhenis

It should be noted that increase in protein content is not specific for all mutant lines and depends on initial genotype, as could be seen from the Table 2. For example, under the influence of Tween 20 surfactant at a concentration of 1% on Shagala variety (club-shaped spike), the content of dissolved protein was reduced by 28% in compare with the control (0.137 mg/ml and 0.188 mg/ml, correspondingly).

Changes in the activities of enzymes, involved in the processes of metabolism, is one of the most reliable criteria for detection of alterations in genetic apparatus under the influence of mutagens. Therefore, study of activity of key enzymes of nitrogen and energy metabolism processes in mutant genotypes in compare with the original varieties has been of particular interest.

The study on enzymatic activity provides an opportunity to esteem the intensity of metabolism of the plant organism, and more reliably estimate the vitality of obtained mutant genotypes.

Decline in enzymatic activity of GDH, ADH and EC MDG - GOAT was shown at all mutant forms, except mutant line 1. Reduced activity of GDH enzyme is a favorable anti-stress factor, since this enzyme promotes excretion of toxic ammonia, destroying biomembranes.

The overall decline in the enzymatic activity of ADH indicates suppression of anaerobic respiration. Conversely, during study of enzymatic activity of MDG, involved in reactions of energy metabolism at all mutant lines of Shagala variety showed its rise from 4 to 109%. Because the enzyme MDG participates in the process of respiration, increase in its activity indicates an improvement of energy processes.

Study of activity of key enzymes of nitrogen and energy metabolism showed that mutants, induced by surfactants, had a significantly higher or lower enzymatic activity in comparison with the original varieties. This indicated presence of genetically fixed biochemical traits of obtained mutant genotypes. These fixed traits can be used as enzymatic markers for the involvement of mutant lines in practical breeding.

Thus, studied mutant genotypes of wheat induced by surfactants in a concentration of 1%, not only had specific morphological characteristics, but also differ from the original cultivars by their biochemical parameters, such as content of soluble proteins and activity of key enzymes of nitrogen and energy metabolism. This shows prospects for use of studied surfactants at 1% concentration as weak mutagens during creating of initial wheat genotypes.

After treatment of seeds of soft wheat Zhenis and Shagala varieties with surfactants mutant lines with the "hairiness" trait have been obtained, so it was interesting to study the density and size of leaf blade trichomes. Wheat trichomes are single-celled or multicellular formations of epidermis, and refer to simple opaque hairs [12].

Leaf pubescence in wheat is of great biological significance in adaptation to environmental factors.

The study of peculiarities of pubescence morphology, as well as identifying genetic factors, responsible for its formation, allows obtaining varieties, resistant to harsh climatic conditions, as well as immunity to a number of pests [13].

The objects of the study were front flanking leaves of mutant lines, possessing "hairiness" trait, as well as original varieties.

Shagala variety is not a carrier of the "leaf hairiness" trait, and Zhenis - is. Measurement of length of the trichome cells and determination of their density (number of trichomes per unit of area) were performed on the upper and lower leaf blades.

Results of comparative evaluation of the "hairiness" trait disposition in the mutant lines and initial varieties are represented on Figure 1. The results showed that the mutant plants, induced by surfactants, differ from the original varieties by the presence of trichomes and their number per unit of area.

Density of trichomes on the bottom surface of the leaf blade was high as compared with their number on the top of the leaf blade, and was noted to be up to 3 times.

According to the density of trichomes per unit of area, plants contingently divided into groups with a high of 55.5 - 69.8 mm², average - from 45.8 to 52.8 mm² and low - from 22.1 to 38.8 mm² densities were isolated. For example, Line 1, line 3, line 8 varieties of Shagala and the line of 24 varieties Zhenis induced by Triton X-305, Triton X-100 and Tween 65 were classified as plants with a high density of trichomes on the bottom side of the leaf blade.

1 - line 1, 2 - line 2, 3- line 3, 4 - line 4, 5 - line 5, 6 - line 6, 7 - line 7, 8- line 8, 9 - line 9, 10 - line 10, 11 - line 11; 12 - line 23, 13 - line 24.

Fig. 1 - The overall distribution of trichomes on leaf surfaces of mutant lines of wheat varieties Shagala (1-11) and Zhenis (12-13)

The length of the trichomes is a marker sign, according to which selection of plants resistant to harsh climatic conditions of the environment and biotic factors can be carried out. In this connection length of trichomes in the mutant lines of wheat was identified (Table 3).

Table 4 - Length of leaf trichomes of original varieties Shagala, Zhenis and mutants induced by surfactants

According to the length of trichomes mutant lines of wheat were divided into lines with: long (from 0.202 to 0.324 mm) and short (from 0.143 to 0.181 mm) trichomes. Mutant lines 1, 7, 23 and 24 with length of trichomes varying from 0.260 ± 0.04 to 0.324 ± 0.05 mm represent breeding value (Figure 2).

Fig. 2 - The overall distribution of trichomes on leaf surfaces of mutant lines of Shagala wheat variety (х 200)

Conclusions

Thus, for the first time, with the use of surfactants 11 constant mutant genotypes of Shagala variety, and 2 lines of Zhenis variety, have been obtained, which have valuable economic traits, in compare with the original genotypes. The study of obtained mutant genotypes showed that they not only have specific morphological characteristics, but also significantly differ from the initial varieties in their biochemical parameters, such as the content of soluble protein, the activity of key enzymes of energy metabolism and the exchange of glutamate - central amino acid of nitrogen metabolism. All of this suggests great prospects of weak mutagens - surface-active substances use in obtaining valuable genotypes of soft wheat.

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