Yield of crops is determined by many processes originating in the genetic information
of the nucleus, chloroplast and other organelles of the higher plant cell. The
information controls the synthesis of structural proteins and enzymes which
synthesize other cell components. Biotic and a-biotic stresses cause changes
in normal physiological functions of all plants, including economically important
cereals as well. Drought stress, which is a natural stress factor, has the highest
percentage with 26% part when the usable areas on the earth are classified in
view of stress factors (Shiri et al., 2009). Barley
is a crop of major economic importance and also a model species for genetics
and physiology (Koornneef et al., 1997). Barley
is grown under varying agro-climatic situations. It is an important crop grown
worldwide for food, feed and forage. Due to hardy nature, superior nutritional
and medicinal importance barely is being considered as highly needed crop of
present era. It has superior nutritional qualities due to presence of beta-glucan
(an anticholesteral substance), acetylcholine (a substance which nourishes our
nervous system and recover memory loss), easy digestibility (due to low gluten
content) and high lysine, thiamin and riboflavin (Chand
et al., 2008). The barley is an important cereal crop in Azerbaijan.
Drought negatively effects barely growth and development in the world and Azerbaijan
and causes a sharp decrease of barely productivity.
Plant adaptation to unfavorable environmental conditions depend on normal synthesis
of protein. DNA structure and its function have the key role in normal protein
synthesis. DNA-strand consists of different parts, which they have different
structure and functions, Such as labile DNA and stabile DNA. Labile DNA is active
part of DNA and located in euchromatin. This part of DNA has genetics code to
synthesize protein but stable DNA is inactive part of DNA and located in heterochromatin
which it closely connected with histons (Shiri et al.,
Tolerantof crops to stress factors depends on functional state of genome under
stress condition. Genome activity and genetic ordering mechanism is connected
with the structural condition of the DNA (Shiri et al.,
Although, many studies have been done on plants under water deficit, but there
were a few studies on the genetic structure of plants (Aliyev
et al., 2000; Aliyev and Abbasov, 2004; Shiri
et al., 2009). Aliyev and Abbasov (2004) investigated
genome structure under drought and salinity stress. They concluded that the
rate of stabile DNA, labile DNA and RNA was increased in tolerant variety under
stress condition but in susceptible variety; the rate of those was decreased
under stress condition. The similar results were obtained by Shiri
et al. (2009) in maize hybrids under drought stress conditions.
Phytohormones are among organic molecules which markedly influence many metabolic
reactions of plants when present in only minor quantities. In all the crops,
phytohormones mainly play important roles in transmission of information within
a plant. Thus as basis for cultivation and breeding of crops, the elucidation
of physiological effects of phytohormones is essential (Kojima,
Aliyev et al. (2000) and Pustoraytova
(1990) stated that GA3 has an important role in the adaptation
and resistance of plants to water deficit.
The aim of this study is to investigate structural and functional changes caused by stress factors in barley genome chromatin, as well as, to gain some knowledge about the mechanism of this effect.
MATERIALS AND METHODS
This study was carried out on 12 barley (H. nutans Schubl.) varieties named as Huseyn-1, Arpa-47, Arpa-84, Nutans-303, Nutans-67/91, Arpa-43, Jalilabad-19, Arpa-77, Nutans-86-35/18, Arpa-59, N-Selection and Nutans-80-34/14. This experiment was done at Genetic Resources Institute of Azerbaijan National Academy of Sciences in 2008.
For classifying barley varieties to two groups (drought tolerant and drought susceptible groups), seed germination ability was used under artificial water stress condition in laboratory condition. Artificial water stress was created with sucrose solution at 10 atm. Then, one variety was chosen from each group (drought tolerant and drought susceptible groups). Then, for studying genomic structure, the stabile DNA, labile DNA, residual DNA and RNA were extracted from selected variety. 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 into two equal groups after 5 days. The first was irrigated with 100 mL of distilled H2O and the second with 100 mL of PEG (60 g PEG/L d H2O: 05 atm.) twice a day at 12 h intervals. The PEG was used to create drought stress. At the end of 48 h, for extraction of nucleic acids, 2 g fresh leaves of seedlings were collected from each group. The rest of the seedlings irrigated with dH2O were kept as a control group and the rest of the seedlings irrigated with PEG were divided into two groups. The first group was irrigated with 100 mL of dH2O and the second group with 100 mL of gibberellic acid (GA3) plus Kinetin (50+50 mg L-1) twice a day at 12 h intervals over four days. Leaf samples from these groups were randomly collected after 96 h the onset of stress (72 h 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 Konarev and Tyuterev
(1970) and Alekseev (1973) methods. Nuclear nucleic
acids were extracted by the gradual fractionation method (Konarev
and Tyuterev, 1970). 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). Stable chromatin, labile chromatin, residual
chromatin and RNA content was determined by ultraviolet absorbency difference
at 270 and 290 nm wave-length as mg mL-1
according to Konarev and Tyuterev (1970).
RESULTS AND DISCUSSION
The studied variety divided to three groups based on germination ability under artificial water deficit condition according to cluster analysis by ward method (Fig. 1). Discriminate function analysis confirmed the classifying correctness in cluster analysis (data not shown). According to cluster analysis based on germination ability of barely varieties under artificial drought stress and control condition, Huseyn-1, Arpa-84, Arpa-83 and Arpa-47 had the highest rate of germination under drought stress condition and considered as drought tolerant varieties. Whereas, N-Selection, Arpa-59 and Nutans-80-34/14 had the least rate of germination under drought stress condition and considered as drought susceptible varieties and the rest of varieties had moderate rate of germination under drought stress condition (Fig. 1).
The result of on nucleic acids content showed that in drought tolerant variety,
Huseyn-1, the amount of labile DNA, which is the active portion, increased 21.9%
under drought condition after 24 h in comparison to control but in drought susceptible
variety, N-Selection, the amount of labile DNA (free DNA) decreased 23.81% under
drought condition in comparison to control. In case of RNA content, in drought
tolerant variety, Huseyn-1, the amount of that increased 16.5% under drought
condition in comparison to control but in drought susceptible variety, N-Selection,
the amount of RNA decreased 8.13% under drought condition in comparison to control
(Fig. 2a, b). The rate of RNA and DNA in
a cell shows the transcription amount of DNA (Smith and Grierson,
1982). Aliyev and Abbasov (2004) investigated genome
structure under drought and salinity stress. They concluded that the rate of
stabile DNA, labile DNA and RNA was increased in tolerant variety under stress
condition but in susceptible variety; the rate of those was decreased under
stress condition. The similar results were obtained by Shiri
et al. (2009) in maize hybrids under drought stress conditions. Transformation
from stabile DNA to labile DNA is possible within cell during morphogenetic
processes which it could be affected by genetic and environmental stress (Aliyev
et al., 2000). Therefore, the ability of tolerant varieties in suffering
stress is because of their potential to increase the rate of labile DNA and
RNA content, thereby increasing protein synthesis.
In drought tolerant variety, Huseyn-1, the amount of stable DNA (DNA bound
loosely to histones), residual DNA and total DNA increased 13.8, 29 and 19.9%,
respectively under drought condition in comparison to control but in drought
susceptible variety, N-Selection, the amount of those decreased 36.4, 34 and
30.45%, respectively under drought condition in comparison to control (Fig.
||Dendrogram for 12 barley varieties based on germination ability
under drought stress created saccharose solution with 10 atmosphere pressure
||Variation (%) of RNA and DNA fractions in drought stress condition
(created with PEG) in comparison to control (a) in drought susceptible variety
(N-Selection) and (b) in drought tolerant variety (Huseyn-1)
In the seedlings applied Gib+Kin phytohormones complex following PEG application,
the DNA content increased in comparison to the water applied following PEG seedlings.
The increasing in the amount of labile, stable DNA , residual DNA and RNA was
38.5, 46.9 , 50.9 and 30.1%, respectively in drought tolerant variety and was
21.1, 20.9 , 34.8 and 37.9%, respectively in drought susceptible variety (Fig.
3a, b). The similar results were obtained by Aliyev
et al. (2000). Thus, it is suggested that Gib+Kin complex activates
the synthesis of nucleic acids and strengthens the resistance of plants to water
deficit. In the study carried out by Shang et al.
(1994), it was determined that the foliar application of plant growth regulator
increases the resistance of wheat seedlings to the water deficit. This increase
can be explained by the increase in the active portion of chromatin DNA. These
observations are interpreted as phytohormones molecules (Gib+Kin complex) bind
to histones chemically and remove the repressing effect of histones on genes.
||Variation (%) of RNA and DNA fractions in application of Gib+Kin
phytohormones complex following PEG in comparison to irrigation with dH2O
following PEG (a) in drought susceptible variety (N-Selection) and (b) in
drought tolerant variety (Huseyn-1)
After application of phytohormones (Gib+Kin) complex following drought stress,
genome activation increased both in tolerant and susceptible varieties, resulting
in the increase of RNA and DNA fractions. Therefore, Gib+Kin complex application
alleviates drought stress effect by activating the synthesis of nucleic acids
and allows the maintenance of normal functions of plant cells.
Results obtained from this investigation showed that changes occurred in genome structure and functioning can be accepted as stress resistance indices of plants and can be used in explanation of plant resistance and molecular-genetic mechanisms of phytohormone influence.
Thanks to the Prof Ramiz Tagi Aliyev for his kind support, help and suggestions during this research work.