GLUTAMINE SYNTHETASE
ACTIVITY AND FREE AMMONIA ACCUMULATION IN PEA (PISUM
SATIVUM L.) ROOTS AND NODULES AS FUNCTION OF TEMPERATURE
IMPACT
Anatolii K. Glyanko, Nina V. Mironova, Galina G. Vasilieva
Siberian Institute
Plant Physiology and Biochemistry SD RAS
POB 1243, 6640333, Irkutsk,
Russia, e-mail – ustaft@sifibr.irk.ru
Abstract:
The
research was focused on the impact of different temperature (8 and 22îÑ) in the root zone on the activity of one of the
major enzymes of ammonia assimilation – glutamine synthetase (GS) and free
ammonia accumulation in the pea roots and nodules tissues (Pisum sativum L.,
cv. Marat) in the “blooming initiation” phase. GS was proven to act as a basic
enzyme of bacteroid NH3 assimilation at low temperature in the root
zone (8îÑ), whereas at optimal temperature (22îÑ) other enzymes
associating ammonia are likely to participate in this process.
Key Words: Glutamine synthetase; free ammonia; root and nodules
pea; low above-zero temperature
Low
above-zero temperature in the legumes root zone is known to negatively affect
symbiotic nitrogen fixation [1, 2]. According to some authors this is due to
the disturbance of bacteroid ammonia outflux and assimilation, this substance
being the first stable product of nitrogen fixation of legumes [3].
Nevertheless, this issue remains unclear up to now. In compliance with the
presently dominating view, NH3
diffuses through peribacteroid membrane (PBM) separating bacteroid and
plant (noninfected) nodule tissues (4, 5].
NH3 concentration gradient on one and the other side of PBM
is created thanks to high activity of both nitrogenase and glutamine synthetase
(GS), which catalyzes glutamic acid ammination reaction with glutamine
production [6]. Glutamin enters in the transammination reaction forming two
glutamate molecules, with glutamate synthase acting as catalyst, the former
along with GS being highly active in nodule cytosol [7]. Enzymes of bacteroid
NH3 assimilation are localized in noninfected nodule part; enzymes
either show insignificant activity in the bacteroids or are absent [4]. The
present work was aimed at the evaluation of low above-zero temperature impact
in the pea root zone on the activity of major enzyme of bacteroid NH3
assimilation - GS and accumulation of free ammonia in root and nodule tissues.
Materials and Methods
The
object of study was presented by pea plants (ñv Marat) grown in enamel vegetation vessels with the
capacity of 5 kg of air-dry sand soil with the addition of Gelrigel nutrient
mixture [8]. To inoculate seeds
commercial bacterial compound “Rhizotorfin” containing active form of nodule
bacteria Rhizobium leguminosarum bv. viceae
(strain ÑIAM 1026)
[9] was introduced in the soil. Nitrogen content in nutrient medium (in the
form of Ca(NO3)2 ) amounted to 80 mg-1 kg-1 of soil. The plants (5 in each vessel) were
grown in the Siberian phytotron chamber (Irkutsk, Russia) illuminated by xenon
lamps with radiation intensity 300 W/m2 and photoperiod 16/8 h (day/night). The
plants were irrigated up to 60% of
complete soil humidity ratio. Different temperature in plants root zone (8±1
and 22±1îÑ) at the same air temperature for both variants (22îÑ) was created by introducing flowing water of certain
temperature.
GS
activity was determined in pea roots and nodules samples fixed by liquid
nitrogen [10]. Plant samples frozen in liquid nitrogen (1.5 g) were homogenized
in 6 ml of 0.1 Ì of tris-buffer (ðÍ 7.8) with the addition of EDTA (0.5 mM), polyamid
for phenol association (100 mg) and a-mercaptoethanol (0.05 ml). Homogenate was filtered through
double layer of kapron tissue and centrifuged at 6000g during 15 minutes;
supernatant was centrifuged at 18000g during 30 minutes (in the process of
further nodules analysis this fraction was marked as cytosolic). Resulting
supernatant fluid was used for assay of enzymes activity. Activity of glutamate
dehydrogenase was determined according to the protocol (11]. All the operations
were performed at 4îÑ. The content of free ammonia was determined in
supernatant of roots and nodules fractions after proteins precipitation [12];
protein content was measured using amid-black dye [13]. Nodules sample (0.5 g)
was incubed within 1 hour in 5 mM solution of α-måthionine sulfoximine (ÌSÎ) (specific GS inhibitor) (Sigma, USA). The experiments were reproduced 3-4 times.
The results are presented as Ì ± S.
Results and Discussion
Free ammonia content in pea plants nodules in the phase of
“blooming initiation” was the same both at low and optimal temperatures in the
root zone (Table 1). In pea roots free ammonia content was 4.4 times higher at
low temperature than at optimal temperature. At low temperature ammonia
concentration in the roots was 168% higher than in the nodules, and at optimal
temperature of plants growth it was accordingly 2.7 times lower. GS activity in
the nodules proved to be twice as high both at optimal and at low temperatures
in the root zone (Table 1).
At low temperature the activity of glutamate
dehydrogenase, another enzyme of ammonia assimilation, more than doubled in the
roots - 0.384 and 0.151 mM NADH2 /g-1 of fresh tissue min-1.
This agrees with the other data ([14, 15]. Consequently, it may be presumed
that low temperature in the roots zone causes dusturbance of the processes
connected with assimilation of both exogenous and, possibly, endogenous
nitrogen.
To check this supposition we conducted tests
using α -
methionine sulfoximine (ÌSÎ),
specific inhibitor of GS activity. As tests results showed, incubation of
nodules separated from roots in ÌSÎ solution contributed to the accumulation of
free ammonia in nodules cytosol fraction of the plants growing at low
temperature and brought about decrease of ammonia concentration as compared to
control variant (nodules incubation on water) at optimal temperature of plants
growing (Table 2). ÌSÎ inhibited GS activity by 35 - 45%, and to a higher degree
at low temperature.
Thus, the
pattern of MSO impact on free ammonia accumulation in the nodules varies
depending on the temperature of plants growth medium. Reduction of GS activity
in the nodules due to MSO influence should lead to ammonia accumulation,
however, it accumulates only in the nodules of plants grown at low medium
temperature. At the same time at optimal temperature free ammonia concentration
not only fails to grow under MSO impact, but even drops as compared to control.
Presumably, GS is not the only enzyme responsible for bacteroid NH3 assimilation in this case. In
other words, there are other possible ways of NH3 assimilation, for
instance, through the reactions catalyzed by asparagine synthetase ot alanine
dehydrogenase. This assumption is confirmed by the results of the research
conducted by Knight and Langston-Unkeffer [16], who indicates that tabtoxinine-β-lactam, specific inhibitor
of root and nodule GS produced by tobacco pathogen Pseudomonas syringae pv. tabaci, along with more than 50%
inhibition of nodule GS activity in alfalfa, contributes to intense plants
growth, increase of nitrogenase activity and total nitrogen contents in the
plants. In this connection the authors assume that there exist alternative ways
if bacteroid NH3 assimilation in alfalfa nodules (apart from
GS-way).
Other
data confirm that NH3 might assimilate immediately in bacteroids.
Thus, according to Waters et al. ([17] ammonia formed in soy plants bacteroids
as a result of nitrogen-fixation, assimilates with the help of alanine
dehydrogenase forming alanin, which is a major transport form of fixed nitrogen
via PMB in the nodule cytosol. These data supported to be true by researches
Allaway et al. [18], which have proved sythesis of alanine in isolated of
bacteroids nodules of pea. In further these authors [19] have shown an
possibility of an exchange amino acids between bacteroid and vegetative parts
of nodule of pea. Therefore, at low temperature GS is likely to be the main
enzyme participating in utilization of bacteroid NH3. Ammonia accumulation supposedly takes place
in these conditions as a result of reduction of alanine dehydrogenase and
asparagine dehyrogenase activity. Increase of ammonia concentration in intact
plants roots at low temperature apparently happens due to absorption and
assimilation of exogenous nitrogen. Due to the fact that GS activity in the
roots does not reduce significantly at low temperature (as compared to optimal
temperature), and, besides, glutamate dehydrogenase activity increases, the
observed free ammonia accumulation probably takes place at the expense of
nitrate nitrogen uptake from nutrient medium and their intense reduction in the
roots plants of pea ”blooming initiation” phase [20].
Thus, it may be concluded that at low
temperature (8îÑ) in pea plants roots zone GS acts as a major enzyme of
bacteroid NH3 assimilation,
and at optimal medium temperature (22îÑ) there possibly function other enzymes participating
in the assimilation of nitrogen fixed by nodule bacteria.
Literature
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Table 1. Free
ammonia concentration and GS activity in pea nodules and roots (cv. Marat) as
function of temperature in the roots zone (“bloming initiation”)
Temperature
in root zone, o C
|
Roots
|
Nodules
|
Concentration
of N-NH4+
/ m -1 g –1 fresh tissue
|
22
± 1
|
46,5 ± 18,2
|
126,6
± 10,1
|
8
±1
|
204,8 ± 17,6
|
121,3 ± 15,6
|
GS
activity / µM -1 γ-glutamilhydroxamate mg-1 of
proteim 20 min-
|
22
± 1
|
2,73
± 0,73
|
6,00
± 1,73
|
8
± 1
|
2,01
± 0,82
|
6,61
± 1,66
|
Table 2. Free ammonia concentration and GS activity in pea nodules and
roots (cv. Marat) as a function of temperature in the roots zone and incubation
of MSO solution and H2O ("blooming initiation"phase)
|
Temperature
in root zone, oC
|
Nodules
incubation on water
|
Nodules incubation on MSO solution (5 mM)
|
|
Concentration
of N-NH4+ / µ -1 g –1 fresh
tissue
|
|
22
± 1
|
138,7 ± 15,1
|
83,3
± 5,6
|
|
8
± 1
|
156,4
± 5,8
|
224,7
± 20,1
|
|
GS activity /
µM –1 γ-glutamilhydroxamate mg –1 of protein 20
min -1
|
|
22
± 1
|
3,95 ± 0,63
|
2,59 ± 0,46
|
|
8 ± 1
|
3,65
± 0,55
|
1,99
± 0,39
|
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