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Changes in genome structure of wheat varieties caused by drought and salt stress and effects of phytohormones on these changes

Ramiz Aliyev and Mehraj Abbasov1

1Azerbaijan National Academy of Sciences Genetic Resources Institute, Baku, Azerbaijan. mehrac777@yahoo.com

Abstract

In this study, five durum wheat varieties (T.durum Desf.) and five bread wheat varieties (T. aestivum L.) were treated with drought and salt stress. RNA and DNA changes of the most susceptible and most resistant variety of each species was then studied in detail. It was observed that the amount of RNA and the fractions of DNA changed after stress . Increases in some fractions appeared to be positively associated with resistance in durum wheat , on the other hand they had a negative effect on the bread wheat varieties. The stresses caused changes on the chromatin structure of the durum wheat genome suggesting increased active or labile DNA and more transcription in the resistant variety. The combination of GA and Kinetin hormones applied after stress increased the nucleic acid repair processes in both bread and durum wheat varieties.

Key Word

Wheat, plant resistance, stress factors, RNA, Fractions of DNA.

Introduction

The stress factors especially drought and salinity negatively effect plant growth and development. They cause a sharp decrease of plants productivity.To achieve high output of agricultural crops under stress, stress resistant varieties are needed. In order to increase stress resistance, it is necessary to study physical and chemical changes in varieties of differing susceptibility caused by the stress, including changes at the molecular genetic level.

Crop resistance to stress factors is connected with their capacity to synthetize RNA and stress resistance proteins, which can arise by the activation of key genes. Dubcovsky et al (1994) have compared the genetic system of salt susceptible wheat and salt resistant Lophopyrum elongatum under early salt stress . They noted that Lophopyrum Elongatum showed a higher level of mRNA than the wheat under the salt stress (and it caused on the result of the changes in gene ordering, what you mean by ordering is unclear). Other studies on the effect of salt have also showed changes in the mRNA complex ( Zahao et al (1989). Genome activity and genetic ordering mechanism is connected with the structural condition of the DNA. The aim of this study is to detail structural and functional changes caused by stress factors in durum and bread wheat genome chromatin, as well to gain some knowledge about the mechanism of this effect.

Materials and Methods

This study was carried out in 5 durum wheat (T.durum desf) and 5 bread wheat (T.aestivum L.) varieties. Their drought resistance was evaluated by the germination ability of their seeds in laboratory conditions at 10 atm.(for durum wheat) and 16 atm.(for bread wheat) osmotic pressure in saccharose solution (Oleynikova et al , 1976). For measuring salt resistance germination in 0.2M NaCl solution was used.

For studying genomic structure and activities, a germination susceptible and a resistant variety was chosen each from durum and bread wheat, and artificial water deficit and salt stress created. For the former PEG-3000 was used (Grodingsky and Osipov, 1973) and the latter NaCl. The seeds were kept in distilled water (dH2O) overnight and germinated in plastic pots (20 cm in diameter) containing air-dried greenhouse soil under natural light at room temperature. Pots containing seedlings were divided in two equal groups after five days. The first was irrigated with 100 ml of distilled H2O and the second with 100 ml of PEG (60g PEG/I d H2O: 05 atm.) twice a day at 12 hour intervals. At the end of 24 hours, 2 g fresh leaves of seedlings were collected from each group in order for genomic nucleic acids to be extracted. The rest of the seedlings irrigated with d H2O were kept as a control group, and the rest of the seedlings irrigated with PEG and with NaCl were each divided in two groups. The first group was irrigated with 100 ml of d H2O and the second group with 100 ml of giberellic acid (GA3) plus Kinetin (50 mg/l + 50mg/l) twice a day at 12-hour intervals over four days. Leaf samples from these groups were randomly collected after 96 hours the onset of stress (72 hours after the relief of stress). To reduce sample variation, all measurements were performed on the second and third leaves of seedlings and samples were collected in four replicates.

Total cell DNA and RNA were isolated by the procedures described below. Nuclear nucleic acids were extracted by the gradual fractionation method. The gradual application of varying ionic power forms the basis of this method, allowing the separation of labile chromatin DNA (free DNA), stable chromatin DNA (DNA bound loosely to histones) and residual chromatin DNA (DNA bound strongly to histones) (Alekseyev 1973; Ovchinnicova and Yocovlev, 1976). Amounts of the nucleic acid were estimated as mg/ml by spectrophotometer with 270 and 290 nm wave-length absorption spectrum (Konaryev and Tyuteryev, 1970).

Results and Discussion

The degree of resistance to salt stress and to drought stress of the wheat varieties studied in the first stage of our investigation are shown in Figure 1.

Figure 1. The germination per cent of the durum and bread wheat varieties in 0.2 M NaCl solution (Salt), and as well, 10 atm (durum wheat) and 16 atm (bread wheat) in saccharose solution (Drought).

Durum wheat (T.durum Desf : varieties: 1-Hordeiforme 41/97, 2-Melanopus 12/97, 3-Lecumelan 2/97, 4-Leucurum Cəfəri, 5-Leucurum Şərq

Bread wheat (T.aestivum L): varieties: 6-Graecum Grgənə, 7-Ferrugineum 254, 8-Lutescens 6709, 9-Erythrospermum 1710, 10-Ferrugineum 7521.

As is seen in Figure 1, amongst durum varieties Leucurum Cəfəri and Leucurum Şərg are the most resistant to salinity; Hordeiforme 41/97 and Melonopus 12/97 are moderately resistant, and Leucumelan 2/97 is the least resistant to salinity. For drought, Leucurum Cəfəri and Melanopus 12/97 are the most resistant to drought and Lecumelan 2/97 the most susceptible. For the bread wheat varieties, Lutescens 6709 is the most susceptible to salt and drought, and Ferrugineum 7521 the least.

In order to detail changes in chromatin structure caused by stress factors, we chose for durum varieties, Leucurum Cəfəri as resistant to salinity and drought, and Leucomelan 2/97 as susceptible. For bread wheat varieties we used Ferrugineum 7521 , as the most resistant to stress factors and Lutescens 6709 the most susceptible. The results obtained are shown in Tables 1 and 2.

For the durum varieties ( Table1) it seems thatafter 24 hours of salt stressing in susceptible Leucomelan 2/97 a reduction in DNA fractions has occurred and the total amount of DNA has also been reduced. In particular the reduction is seen in the RNA and the labile DNA. In contrast, resistant Leucurum Cəfəri shows an increased amount of all DNA fractions after 24 hours, at the same time it also has more RNA (+ 67%) and labile DNA. RNA and labile DNA appear to be correlated. Perhaps in Leucomelan 2/97 the lower transcription velocity is associated with reduced the drought resistance, and in Leucurum Cefari, increased transcription velocity associated with greater resistance.

At 72 hours after stress, there were significant increases of RNA, DNA fractions and total DNA in resistant and susceptible varieties as a result of giving GA+Kinetin hormones. This shows the positive effects of GA+Kinetin hormones on the structural and functional state of genome, as well their effect on increasing the velocity of repair processes.

Table 1. Durum wheat varieties: effect of salt and drought (PEG) stress, without and with GA+Kinetin, on RNA and DNA fractions (mg/100 g of fresh weight).

Wheat variety
and treatment

RNA

DNA fractions

Total
DNA

Labile

Stable

Residual

Leucomelan2/97 (susceptible) 24 hours after the stress

Control

19.07+0

4.50+0.20

2.92+0.20

0.80+0.12

8.22

Salt

18.27+0.51

4.0+0.16

2.63+0.17

0.48+0.03

7.11

PEG

18.78+0.93

3.53+0.18

1.97+0.25

0.56+0.12

6.06

72 hours after the stress

Salt+water

17.62+0.41

7.84+0.22

5.63+0.22

0.90+0.08

14.37

Salt+GA+Kin

26.54+1.45

11.49+0.19

6.66+0.17

1.14+0.15

19.23

PEG+water

14.99+1.38

6.71+0.17

4.37+0.24

0.49+0.05

11.57

PEG+GA+Kin

22.18+2.28

7.90+0.22

4.45+0.20

0.51+0.01

12.86

Leucurum Cəfəri (resistant) 24 hours after the stress

 

Control

13.92+1.07

6.88+0.23

4.43+0.14

0.52+0.11

11.83

Salt

18.75+0.75

7.62+0.22

6.02+0.11

0.56+0.01

14.20

PEG

23.35+1.07

8.38+0.12

4.18+0.12

0.92+0.16

13.48

72 hours after the stress

 

Salt+water

22.81+0.96

5.72+0.22

2.63+0.21

0.90+0.08

9.25

Salt+GA+Kin

28.92+0.42

7.11+0.27

3.96+0.18

1.22+0.04

12.02

PEG+Water

13.50+1.50

7.18+0.14

3.38+0.19

0.74+0.07

11.30

PEG+GA+Kin

18.53+0.11

9.40+0.21

5.06+0.16

1.07+0.08

15.53

Though the changes caused by stress factors in the bread wheat varieties generally are the same as with the durum wheat varieties, they differ from each other according to several characteristics (Table 2). The most significant difference is the absence of strong correlation between labile DNA and RNA amounts. While the susceptible bread wheat variety, Lutescens 6709, in control group the labile DNA number is 8.52 mg/100g of fresh weight, RNA amount is 18.24, in the salt stress group these values are 5.85 and 24.72, respectively. The significant reduction of labile DNA number in salt stress group wasn’t associated with a reduction in the RNA synthesis, in fact it increased. A similar results is seen in the resistant Ferruguneum 7521 variety. The labile DNA amount in the control, salt and PEG groups of this bread wheat are very close to each other. However, RNA amount is doubled in the salt stress group, as well as significantly increased also in the PEG group. This situation shows a greater effect of stress factors, especially of salt stress, on the functional state of the genome in bread wheat i.e. DNA’s transcription velocity or gene expression is greater. A significant increase of RNA in susceptible and resistant wheat varieties as well an increase of total DNA number has occurred in GA+Kin group after the stress. According to these results durum and bread wheat varieties have shown differences as well as similarities. Consequently, it demonstrates a reduction of stress effects with the GA+Kin hormonal combination, especially in bread wheat, and an increase in the velocity of genome repair process. The similar results have also obtained in other studies.

Table 2. Bread wheat varieties: effect of salt and drought (PEG) stress without and with Ga + Kinetin, on RNA and DNA fractions (100 mg of fresh weight)

Wheat varieties
and Treatment

RNA

DNA fractions

Total
DNA

Labile

Stable

Residual

Lutescens 6709 (susceptible) 24 hours after the stress

Control

18.24+0.83

8.52+0

7.43+0.16

0.85+0.07

16.80

Salt

24.72+0.79

5.85+0.12

8.74+0.09

1.29+0.08

15.88

PEG

22.07+0.72

8.81+0.10

7.64+0.16

1.13+0.06

17.58

72 hours after the stress

salt+water

26.43+1.34

7.97+0.23

3.07+0.23

1.10+0.11

12.14

salt+GA+Kin

32.97+1.66

9.27+0.18

3.96+0.17

1.18+0.10

14.41

PEG+water

31.20+1.97

7.92+0.16

6.49+0.25

1.11+0.10

15.52

PEG+GA+Kin

46.24+1.66

11.24+0.13

6.97+0.09

1.29+0.05

19.50

Ferruguneum7521 (resistant) 24 hours after the stress

Control

22.80+0.62

4.92+0.30

8.16+0.16

0.54+0.04

13.62

Salt

44.16+1.03

5.16+0.16

13.48+0.31

0.49+0.01

18.13

PEG

28.09+2.7

5.21+0.21

11.36+0.14

0.71+0

17.28

72 hours after the stress

Salt+water

16.53+1.35

4.45+0.20

9.68+0.38

0.21+0.04

8.34

Salt+GA+Kin

20.77+0.62

5.72+0.17

5.80+0.20

0.39+0.05

11.91

PEG+water

18.70+1.03

3.59+0.11

3.36+0.21

0.28+0.03

7.23

PEG+GA+Kin

19.50+0.42

5.29+0.20

5.41+0.39

0.41+0.03

11.11

References

Alekseev V. G. The heterogeneity of DNA and the activity of the genome in wheat (Triticum aestivum L.) seedlings. Bulletin of Applied Botany. Genetic and Plant Breeding. 52 (1):46-56, 1973.

Dubcovsky J, Galvez A.F and Dvorak J. (1994) Comparison of the genetic organization of the early salt stress response gene system in salt-tolerant Lophopyrum elangatum and salt-sensitive wheat. Theoretical and Applied Genetics87: 957-964.

Grodingsky, A.M., Grodingsky D.M., General information about plant physiology, second edition, Naukova Duma Press., Kiev, (1973), (Russian)

Konarev V. G. and Tyuterev S. L., Techniques on the Cytochemistry and Biochemistry of Nucleic Acids, first edition (Russian). Leningrad. 1970.

Oleynikova, T.V. and Osipov, Y.F., “Wheat and barley species in fluid of high osmotic pressure sacharose druablity evaluation of maise species hybrids’ seeds according to their growth extend. Evaluation methods of crops’ durability in unfavourable condition” 23-31, Leningrad, (1976), (Russian).

Ovchinnicova M. F and Yocovlev A.P Complementation of choloroplast of maize (Zea mays L) Agricultural Biology, 11 (5): 675-679, 1976.

ztrk, M., Gemici, M., zdemir, F., Keyii, N., “The role of crop hormons in salt stress reduction during seed growth,” Ege Uni.Botanic Ana bilim dali S.8, (1994) (Turkish)

Ramiz T. Aliyev, Kamil Joshkunchelebi, Osman Beyazoglu, Maize (Zea mays L.) Changes Caused by Gibbellic Acid on the genetic Systems of Maize (Zea mays L.) Seedlings. (Turkish).

Ramiz Aliyev, Kamil Coshkunchelebi, Osman Beyazoglu and Sefika I. Hacieva. Effect of Giberellic Acid on the Nucleic Acids Content in Wheat Seedlings (T.aestivum L.) Grown under Water Feficit/ Pakistan Journal of Biological Sciences,3, (1): 24-26, (2000). (Pakistan).

Zahao Zhifan, W.Heysez James, J Borhert Hars, and Cell Physiol. Gene Expression In Suspension Culture Cells of the Halophyte Sistichlis Spicata During Adaptation To high Salt, N:6,-C, 861-867.,(1989).

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