THE ACTIVITY OF ENZYMES ASSOCIATED WITH ASPARTATE
SYNTHESIS OF WHEAT AT BIOTIC AND ABIOTIC STRESS
CONDITIONS
Активности ферментов связанных с синтезом аспартата пшеницы при
биотических и абиотических стрессовых условиях
Zh.S. Kudiyarova, Zh.T. Lesova, N. Muttucumaru, N.G. Halford
Almaty Technological University, Almaty, 050012
Rothamsted Research, Harpenden, Herts AL5 2JQ,
zhanar_ks@mail.ru
Abstract: In this work were measured MDH-GOT activities in wheat leaves and
grain, in the case of wheat in varieties with differing degrees of tolerance to drought
stress and rust infections, growing under normal and stress conditions. We have
carried out modifications and optimisations of protein extraction methods for wheat
grain and wheat leaf.
It was measured the activities of MDH-GOT of infected plants of rust-resistant
variety, Naz, and rust-susceptible varieties, Sappo and Alexandria. The activity of
enzyme complex MDH-GOT was measured in non-stressed and stressed wheat seeds
during different developmental stages of varieties Alexandria and Sappo grown in the
glasshouse. It was also measured in irrigated (control) and non-irrigated (drought
stressed) wheat plants of variety Battalion grown in the field. In this study, a high
level of aspartate synthesis of drought stressed plants and rust infected wheat leaves
were shown with MDH-GOT assay.
INTRODUCTION
Asparagine plays a central role in nitrogen storage and transport in plants, due to
it’s a high N:C ratio. This involves accumulation in a range of tissues, particularly
under stress conditions, including conditions where the plant is unable to support a
normal level of protein synthesis.
Asparagine synthetase (EC 6.3.1.1 and EC 6.3.5.4) (AS) catalyses an ATPdependent transfer of ammonia to aspartate yielding asparagine. The reaction requires
magnesium ions and the energy-producing hydrolysis of ATP. Asparagine synthesis
occurs by amidation of aspartate using either glutamine or ammonium as an amino
donor (Larsen et al., 1999).
Asparagine accumulation occurs during normal physiological processes such as
seed germination and nitrogen transport; in addition it appears to be induced by
abiotic and biotic stresses, such as mineral deficiencies, drought, salt, toxic metals
and pathogen attack (Lea et al., 2006). Measurement of AS activity has proven
extremely difficult (Richards and Schuster, 1998; Ireland and Lea, 1999). Problems
associated with assaying AS in plants include the relatively short half life of the
enzyme and its highly regulated activity. The rapid turnover and deactivation
associated with plant AS make extraction of this enzyme in active form difficult, even
in the presence of protectants such as glycerol and thiol-containing compounds (Joy
et al., 1983; Snapp and Vance, 1986). Other difficulties associated with biochemical
studies of AS include the presence of endogenous natural inhibitors (Rognes, 1980)
and relatively high activities of cytoplasmic asparaginase (Hudhes et al., 1997) and
glutamine synthetase (Sieciechowicz et al., 1988), which break asparagine down or
compete for similar substrates.
L-Asparaginase (EC 3.5.1.1) catalyses the hydrolysis of asparagine to aspartic
acid and ammonium, which is subsequently reassimilated for the biosynthesis of other
nitrogen-containing compounds (Lea et al., 2007). Two families of asparaginases are
well characterised: bacterial and plant type asparaginases. Sodek et al. (1980)
demonstrated the presence of two sub-types of plant asparaginases, differing in their
dependence for K+. The potassium-dependent asparaginase appeared to be more
widely distributed in higher plants.
A new potential route for catabolism of glutamate has been identified in wheat
(Koldasova et al., 1999). This involves a new stable enzyme complex consisting of
malate dehydrogenase (MDH) and glutamate oxaloacetate aminotransferase
(GOT/AAT EC 2.6.1.1). This complex (MDH-GOT) catalyses the following
reactions: first, MDH catalyzes the oxidation of malate to oxaloacetate, with
reduction of NAD to NADH; second, oxaloacetate is transaminated with glutamate to
form aspartate and 2-oxoglutarate.
MATERIALS AND METHODS
Protein extraction method for MDH-GOT assay. Wheat leaves or seeds were
homogenized in a chilled mortar with Tris-HCL buffer (50mM), pH 7.5, in ratio 1:4
(w/v) and centrifuged at 10 000 × g for 10 min.
MDH-GOT enzyme assay. MDH-GOT activity was assayed by measuring the initial
rates of reduction of NAD. The increase of absorbance at 340 nm was recorded on
spectrophotometer Ultralight. Crude extracts were used for determination of activity
of MDH-GOT and a reaction mixture contained 1.1 mM NAD, 12 mM malate and
8.7 mM glutamic acid, adjusted to 2 ml with 0.05 M Tris - glycine buffer, pH-8.3.
The reaction was initiated with the addition of 0.2 ml enzyme extract and malate,
measurements were made for 1 minute (A), then glutamate was added and
measurements made during another 1 minute (B). The difference of this two
measurements (B-A) is the activity of MDH-GOT. The units of enzyme activity are in
micromoles of NAD reduced in 1-2 minute per mg of protein in the assay mixture at
25°C.
EC = (∆A340 T0- ∆A340 Tc) MDH - (∆A340 T0- ∆A340 Tc) GOT /ε NADH340x d
µM/mole
Where T= time zero, Tc = time when reaction complete, d = dilution, and ε NADH340
= the extinction coefficient of NADH (0.0062 per µM per ml).
RESULTS AND DISCUSSIONS
It was proposed that the MDH-GOT complex catalyses the main route of
glutamate catabolism without production of toxic ammonia, which would cause
cellular damage, and that the activity of the complex correlates with plant adaptation
to stress conditions and wheat seed viability (Gilmanov et al., 1981). Therefore was
measured the activity of MDH-GOT in plants of rust resistant variety Naz and rust
susceptible varieties Alexandria, Sappo infected with yellow rust (Puccinia
striiformis). Enzyme activity of wheat leaves was measured 14 days after inoculation.
Wheat plants with high MDH-GOT activity had higher tolerance to rust infections
and the stressed plants had the ability to increase the activity of MDH-GOT as a
response to rust infections. Wheat variety Sappo showed more sensitive MDH-GOT
activity, so for further experiments it was used as a rust-susceptible plant (Fig.4).
45
40
35
30
25
20
15
10
5
0
noninfected
rust infected
Alexandria
Sappo
Rust
resistant
Rust
susceptible
Figure 4 Activity of MDH-GOT in leaves of rust-resistant wheat variety Naz and
rust-susceptible varieties Alexandria and Sappo infected with yellow rust (Puccinia
striiformis)
In successfully diseased wheat leaves, MDH-GOT activity was measured every
3-4 days, and this showed gradually increasing activity of MDH-GOT (Fig.5). In
leaves from winter wheat cv. Oakley, Little Knott grown in the field, activity of
MDH-GOT was almost double that of the control non-infected plants. The activity of
the enzyme in rust non-infected plants was not as high as in infected rust-susceptible
plants (Fig.6).
300
250
200
150
Rust infected
100
noninfected
50
0
13.07.2011 18.07.2011 22.07.2011
Figure 5 Changes of activity of MDH-GOT in leaves of
rust-susceptible wheat infected with yellow rust.
Activity of MDh-GOT uM/mg
300.00
250.00
200.00
150.00
100.00
50.00
0.00
noninfected
rust infected
Figure 6 Activity of MDH-GOT of winter wheat leaf cv. Oakley, Little Knott
Activity of EC MDH-GOT µM/mg
Asparagine is the major transport compound in the xylem from the root to the
leaves and in the phloem from the leaves to the developing seeds in a range of plants
(Lea et al., 2007). Activities of MDH-GOT were measured in leaves and seeds under
drought stress at different developmental stages of wheat. In irrigated and nonirrigated wheat plants there wasn’t a big difference, suggesting that the plants were
not severely stressed. Activity in both sets of plants increases in the stems after the
pre-anthesis stage, when assimilates would be going to the development of seeds
(Fig. 7).
The activities of MDH-GOT in non-stressed seeds which were grown in a
glasshouse showed low enzyme activities (Fig. 8), while in drought-stressed seeds
activity increased sharply during milky stages (Fig. 9).
700
600
500
400
300
200
100
0
Irrigated plants
leaf
Irrigated plants
stem
Non irrigated
plants leaf
Non irrigated
plants stem
Figure 7 Activity of MDH-GOT in leaves and stems of drought-stressed wheat at
different developmental stages
EC MDH-GOT activity, µM/mg
prot.
80
70
60
50
40
Alexandria
30
Sappo
20
10
0
7 days 14 days 1 month 2 months hard seed
old seed old seed
old
old
Figure 8 EC MDH-GOT activities of non-stressed rust susceptible
wheat seeds at different developmental stages
800
700
600
500
400
300
200
100
0
Figure 9 MDH-GOT activities of drought stressed wheat seeds
at different developmental stages
The MDH-GOT activities did not show a big change during most developmental
stages, except that at milky stages the activities were increased and its activity in nonirrigated seeds has been shown to be double that in irrigated seeds, which means the
enzyme complex plays an important role during developing and formation of wheat
seeds under drought stress.
Wheat leaves infected with yellow rust (Puccinia striiformis) in glasshouse and field.
CONCLUSION
It has been established that during abiotic and biotic stresses there is an intensive
accumulation of ammonia in plant cells. It has also been proposed that asparagine
synthetase can use ammonia directly as a substrate (Oaks & Ross, 1984). In this
study, a high level of aspartate synthesis of drought stressed plants and rust infected
wheat leaves were shown with MDH-GOT assay.
In irrigated and non-irrigated wheat plants, there wasn’t a big difference in
vegetative organs, such as leaf and stems. In both sets of plants, the activity
increased in the stems after the pre-anthesis stage, when assimilates would be going
to the development of seeds. During seed development at the milky stage, MDHGOT activity was higher, and drought stressed seeds showed double the activity of
irrigated plant seeds; also MDH-GOT activity was low in non stressed wheat seeds
which were grown in the glasshouse.
Wheat plants with high MDH-GOT activity had higher tolerance to rust
infections and the stressed plants had the ability to increase the activity of MDHGOT as a response to drought and rust infections.
In rust-infected wheat leaves in the glasshouse, MDH-GOT activity was
gradually increasing after 14 days of inoculation and in leaves from winter wheat
grown in the field, activity of MDH-GOT was almost double that of control, noninfected plants.
In conclusion MDH-GOT plays an important role during development and formation
of wheat seeds and the response to drought.
Acknowledgements
This work was supported by Rothamsted International Fellowship at Rothamsted
Research. Authors want to thank Jon West for supplying with rust spores and plants.
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