EFFECT OF DIFFERENT NITROGEN SOURCES AND PLANT GROWTH REGULATORS ON GLUTAMINE SYNTHETASE AND GLUTAMATE SYNTHASE ACTIVITIES OF RADISH COTYLEDONS

EFFECT OF DIFFERENT NITROGEN SOURCES AND PLANT GROWTH REGULATORS ON GLUTAMINE SYNTHETASE AND GLUTAMATE SYNTHASE ACTIVITIES OF RADISH COTYLEDONS screenshot

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46
BULG. J. PLANT PHYSIOL., 2002, 28(3–4), 46–56
EFFECT OF DIFFERENT NITROGEN SOURCES
AND PLANT GROWTH REGULATORS ON GLUTAMINE
SYNTHETASE AND GLUTAMATE SYNTHASE ACTIVITIES
OF RADISH COTYLEDONS

Chitra R. Sood, Sumitra V. Chanda*, Yash dev Singh
Department of Biosciences, Saurashtra University, Rajkot 360 005, India
Received July 25, 2002
Summary. The effect of different nitrogen sources – KNO , NH NO and
3
4
3
NH Cl – on glutamine synthetase and glutamate synthase activities of radish
4
cotyledons (Raphanus sativus L.) in the presence or absence of light was
investigated. The plants were treated with different phytohormones viz.
Kinetin (KN), gibberellic acid (GA) and abscisic acid (ABA). In dark ammonia
supplementation was effective in promoting glutamine synthetase activity in
all the treatments, whereas, KNO was effective only in light in dH O, GA
3
2
and ABA treatments. In dark NH NO promoted the glutamate synthase activity
4
3
in dH O while the activity was more with NH Cl in KN treated seedlings.
2
4
GA had no effect, while low concentration of all nitrogen sources promoted
the activity in ABA treated seedlings. The activity was less in light as compar-
ed to dark grown seedlings Varying levels of NADH-glutamate synthase activ-
ity was discernible in all the treatments and addition of ammonia promoted
this activity to some extent. It is suggested that different energy status of the
seedlings during light/dark or with hormonal treatments may affect the activ-
ity of this enzymes differently. However, it is suggested that changes in Fd-
GOGAT should be studied before any definite conclusions can be drawn.
Keywords: Radish cotyledons, plant growth regulators, nitrogen sources, glut-
amine synthetase, glutamate synthase
Abbreviations: GOGAT – glutamate synthase, GS – glutamine synthetase,
dH O – distilled water, KN – kinetin, GA – gibberellic acid, ABA – abscisic
2
acid
* Corresponding author, e-mail: [email protected]

Effect of different nitrogen sources and plant growth regulators on glutamine . . .
47
Introduction
In plant tissue ammonium can be derived either from NO– reduction, uptake of sup-
3
plied NH+ or photorespiration. An additional internal source of ammonium is the cata-
4
bolism of amino acids and N-transport compounds. Based on enzymological and
inhibitor studies, Miflin and Lea (1976) proposed that the primary pathway for the
assimilatory NH+, in plants, both exogenously and endogenously generated, involves
4
the combined activities of glutamine synthetase (GS; EC 6.3.1.2.) and glutamate syn-
thase (GOGAT; EC 1.4.1.14). Much biochemical and genetic evidence available sup-
ports this conclusion. For example, it has been shown that the potent inhibitors of GS and
GOGAT block NH+ assimilation (Rhodes et al., 1986a). Further, kinetic studies with
4
14N and 15N indicate that glutamine-amide is generally the first and most heavily labeled
product of NH+ assimilation and that glutamine carries a large percentage of the total
4
N-flux of the plant tissue (Rhodes et al., 1986a,b). Further, GS-deficient mutants exhibit
rapid accumulation of free ammonia under photorespiratory conditions (Joy, 1988).
GS catalyses the initial assimilation of NH+ into glutamine while GOGAT, is
4
responsible for its transamidation of glutamine-amidonitrogen to the amino position
of 2-oxoglutarate, to form two moles of glutamate. Because the two enzymes are
dependent on each other for the provision of substrate, their activities constitute a cycle
which has been termed as glutamate synthase cycle. Multiple forms of these two en-
zymes are present in most higher plants. The cytosolic (GS ) and chloroplastic (GS )
1
2
forms of GS show distinct molecular weights, and are encoded by differentially regul-
ated genes in several species (Beckeret. al., 1992; Sakakibara et. al., 1992). Similarly,
two isoforms of GOGAT have been purified and characterized, one dependent on fer-
redoxin as reductant (Fd-GOGAT; EC 1.4.7.1), whereas, the other utilizes NADH
(NADH-GOGAT; EC 1.4.1.4.) (Suzuki et al., 1982).
Studies with barley mutants lacking GS , show that the major role of GS in
2
2
chloroplasts is to deal with the flux of NH+ through the photorespiratory N-cycle (Wal-
4
lsgrove et al., 1987). Similarly, mutants lacking Fd-GOGAT in chloroplasts are also
lethal mutations Similarly, NO– increased the abundance of GS and Fd-GOGAT
3
2
protein in cultured rice cells (Hayakawa et al., 1990).
The assimilation of inorganic N into amino acids, proteins, and other macromolec-
ules requires provision of carbon skeletons. A cell, therefore, is dependent upon the
product of recent photosynthesis or endogenous carbohydrate reserves. It has been
shown that in cells lacking readily metabolizable carbohydrate reserves, NH+ assimila-
4
tion is greatly reduced (Turpin et al., 1991). Many authors reported that NH+ enrich-
4
ment both to higher plants and algae, causes a decrease in the flow of recent photosyn-
thesis to sucrose and starch (Preiss et al., 1985), and that the supply of reducing equiva-
lents and carbon skeleton may regulate NH+ assimilation (Turpin et al., 1988).
4
Considering the aforesaid, in the present paper, changes in the levels of glutamine
synthetase and glutamate synthase activities, in hormone treated radish seedlings

48
Ch. R. Sood et al.
grown with different concentrations of KNO , NH Cl and NH NO in light and dark
3
4
4
3
conditions, were investigated.
Material and Methods
Seeds of radish (Raphanus sativus L) were soaked in dH O for 1 h and germinated in
2
the dark on a wet filter paper (Whatman No.1) for 36 h. Seedlings with radicle length
of approximately 1 cm, were transferred to sieve culture dishes containing N-free nutrient
solution (Doddema and Telkamp, 1979) and phytohormones (KN 20 µM, GA 150 µM,
ABA 40 µM). Half of the dishes were then transferred to light (Ca, 20 µM.s–2.m–1)
and the rest were kept in a dark room at constant temperature (25±2 °C). The seedlings
were allowed to grow under continuous light/dark conditions for 24 h. Seedlings of
equal size were selected and transferred to different concentrations of KNO , NH Cl
3
4
and NH NO (0, 5, 15, 30, 45, 60, and 90 mM), prepared in fresh nutrient solution
4
3
containing different phytohormones. For another 17 h the same hormone-pretreated
seedlings were incubated with different N-forms in light/dark. The enzyme extract
was prepared from the cotyledons of these seedlings for the estimation of both GS
and GOGAT activities.
Enzyme extraction
For the preparation of enzyme extract, required number of cotyledons, from seedlings
of the above described treatments, were separated off and weighed. The chilled material
was homogenized in pre-chilled Tris-HCl extraction buffer (300 mM pH 7.8) contain-
ing ethylenediaminetetraacetic acid (EDTA) (100 mM) and mercaptoethanol (5 mM),
in a cooled mortar at 4 °C in a cold room. The homogenate was first filtered through
4–5 layers of muslin cloth and then centrifuged at 15000×g for 20 min. The resulting
supernatant was passed through a 15 ml column of sephadex G-25 pre-equilibrated
with the same extraction buffer. The desalted preparation was then used for the assay
of the enzymes.
Assay of glutamine synthetase
GS activity was assayed following the modified method of McCormack et al. (1982).
The assay mixture 92.2 ml consisted, of 114 mM imidazole buffer (pH 7.2), 11.4 mM
ATP, 45 mM NH OH:HCl, 45 mM MgSO .7H O and the enzyme extract. After 45 min
2
4
2
of incubation at 30 °C the reaction was terminated by adding FeCl reagent (10% FeCl
3
3
in 0.2 N HCl, 24% TCA and 50% (v/v) HCl mixed in the ratio of 1:1:1). A blank, to
which FeCl was added prior to the addition of enzyme, served as the control. The
3
precipitated protein was removed by centrifugation. The r-glutamyl hydroxamate in
the supernatant was measured at 540 nm. The activity was expressed as nmol glutamyl
hydroxamate produced.h–1.cotyledon–1.

Effect of different nitrogen sources and plant growth regulators on glutamine . . .
49
Assay of NADH-glutamate synthase
NADH-GOGAT activity was estimated by spectrophotometric assay as described by
Chen and Cullimore (1988). The assay mixture in a 3 ml final volume, consisted of
40 mM potassium phosphate buffer (pH 7.5), 10 mM L-glutamine, 10 mM 2-oxogluta-
rate, 0.14 mM NADH and enzyme extract. The control lacked glutamine, NADH and
2-oxoglutarate. Reaction was started with the addition of enzyme. The oxidation of
NADH was observed at 340 nm and the activity is expressed as nmol NADH oxidiz-
ed.h–1.cotyledon–1.
In all the enzyme assays, optimum pH and conditions for linear rate were deter-
mined with respect to substrate concentration and time. The experiments were repeated
at least three times and results of one of the replicate experiment are presented.
Results and discussion
Even in the absence of external NO– or NH+, the cotyledons recorded considerably
3
4
high GS activity, in dark (Fig. 1a). However, upon external addition of NO– and/or
3
NH+, a considerable variation in GS activity was discernible. The concentration-res-
4
ponse curves of the three N-sources used revealed that the addition of increasing con-
centrations of KNO inhibited GS activity while, NH Cl promoted it. The activity was
3
4
intermediate in NH NO treatment. In contrast to this, GS activity in light was signif-
4
3
icantly promoted by all the three nitrogen sources used and maximum promotion was
recorded in KNO and NH NO (Fig. 1b) In higher plants GS functions to assimilate
3
4
3
NH+ generated or mobilized during processes such as seed germination, photorespira-
4
tion, NO– reduction, N fixation and primary NH+ assimilation from the soil (Miflin
2
2
4
and Lea, 1982). The two isoforms of GS have been shown to be localized in different
subcellular compartments (chloroplast and cytosol) (Hirel and Gadal, 1981) and are
differentially present in various organs. Studies on pea with GS cloned genes have
shown that the expression of chloroplastic GS mRNA (GS mRNA) in leaves is regul-
2
ated by light, in a phytochrome mediated fashion (Tingey et al., 1989), and that the
levels of GS mRNA are affected by photorespiratory growth conditions. This suggests
2
a major role of GS in the reassimilation of photorespiratory NH+. In contrast, the GS
2
4
1
protein and transcripts are found at relatively high levels in non green tissue, such as
roots and etiolated shoots, and its role in the generation of glutamine for intracellular
N-transport from cotyledons of germinated seedlings and in N fixing nodules have
2
been highlighted (Coruzzi, 1991). In the present study NH+ supplementation resulted
4
in an increase in GS activity. The similar effects with NH+ on GS activity have also
4
been reported by, Sugiharto and Sugiyama (1992). However, addition of KNO pro-
3
moted GS activity, only in light while in dark it was inhibited.

50
Ch. R. Sood et al.
nmol r-glutamyl hydroxamate produced/h/cotyledon
nmol r-glutamyl hydroxamate produced/h/cotyledon
Concentration, mM
Concentration, mM
Fig.1. Changes in glutamine synthetase activity
Fig.2. Changes in glutamine synthetase activity
with different ambient concentrations of KNO
with different ambient concentrations of KNO
3
3
(X—X), NH Cl (o—o) and NH NO (o—o) in
(X—X), NH Cl (
NO (o—o) in the
4
4
3
4
•—•) and NH4
3
the cotyledons of (a) dark grown and (b) light
cotyledons of (a) dark grown and (b) light grown
grown dH O treated radish seedlings. Vertical
KN treated radish seedlings. Vertical bars rep-
2
bars represent ±SD (wherever bars are absent,
resent ±SD (wherever bars are absent, SD is so
SD is so small that it is within the symbol).
small that it is within the symbol).
Redinbough and Campbell (1993) have shown that GS mRNA transcripts ac-
2
cumulate rapidly and transiently in cells exposed to NO– while NH+ treatment had
3
4
no effects, and suggested that NH+ taken up by the roots is assimilated via GS .
4
1
In the absence of any form of N in the medium, KN treatment promoted GS activ-
ity in light as well as in dark. Supplementing these seedlings with NH Cl significantly
4
promoted GS activity (Fig. 2a) while, addition of KNO decreased the activity. In
3
NH NO supplementation, slight promotion in activity was discernible. In light grown
4
3
KN-treated seedlings, NH NO and NH Cl addition promoted GS activity while,
4
3
4
KNO was not effective (Fig. 2b). Treatment of seedlings with GA also promoted GS
3
activity in the absence of N in the medium. Both NH NO and NH Cl further promot-
4
3
4
ed the activity while, KNO inhibited it (Fig. 3a). Like dH O-control, here also, KNO
3
2
3
supplementation was most effective in light while, NH NO and NH Cl showed pro-
4
3
4
motion only at higher concentrations (Fig. 3b). Abscisic acid promoted GS activity

Effect of different nitrogen sources and plant growth regulators on glutamine . . .
51
nmol r-glutamyl hydroxamate produced/h/cotyledon
nmol r-glutamyl hydroxamate produced/h/cotyledon
Concentration, mM
Concentration, mM
Fig.3. Changes in glutamine synthetase activity
Fig. 4. Changes in glutamine synthetase activity
with different ambient concentrations of KNO
with different ambient concentrations of KNO
3
3
(X—X), NH Cl (
NO (o—o) in
(X—X), NH Cl (
NO (o—o) in
4
•—•) and NH4
3
4
•—•) and NH4
3
the cotyledons of (a) dark grown and (b) light
the cotyledons of (a) dark grown and (b) light
grown GA treated radish seedlings. Vertical bars
grown ABA treated radish seedlings. Vertical
represent ±SD (wherever bars are absent, SD is
bars represent ±SD (wherever bars are absent,
so small that it is within the symbol).
SD is so small that it is within the symbol).
only in light in the absence of N in the medium. In dark, both NH NO and NH Cl
4
3
4
were able to enhance GS activity and KNO inhibited it (Fig. 4a). In light grown
3
seedlings, on the other hand, KNO promoted GS activity which is similar to the
3
promotion recorded in dH O-control and GA grown seedlings (Fig. 4b).
2
From the above mentioned experiments, it was clear that in dark, NH+ supplemen-
4
tation was effective in promoting GS activity while NO– was effective in light. N-
3
assimilation is among the most energy-intensive processes in plants, requiring the
transfer of 2 electrons for NO– converting to NO–, 6 electrons for NO– converting to
3
2
2
NH+ and 2 electrons and 1 ATP per NH+ converting to glutamate. In light, the ATP
4
4
and reductant supply, necessary for these reactions, are generated photochemically
(Anderson and Done, 1977), whereas, the dark-grown plants may divert a significant
proportion of reductant from mitochondrial electron transport (Bloom et al., 1992).
During dark NO– assimilation, shoots of higher plants (Bloom et al., 1989) and algae
3

52
Ch. R. Sood et al.
(Weger and Turpin, 1989) evolved CO sufficiently faster than they consumed O ,
2
2
presumably because the TCA cycle or the OPP pathway catabolized the substrate and
transported some electrons to NO– and NO– rather than to O . These results indicated
3
2
2
that in dark, shoots expend up to 25% of their respiratory energy on N-assimilation
(Bloom et al., 1989), while studies with NH+ assimilation indicated that nearly 14%
4
of carbon catabolism is coupled to NH+ absorption and assimilation (Bloom et al.,
4
1992). The lower activity of GS during KNO supplementation in dark may, therefore,
3
indicate that the availability of ATP and reductant may be limited when compared to
light-grown seedlings. It is only in KN-treated and KNO -fed seedlings that KNO
3
3
was not able to promote GS activity in light (Fig. 4b). In an earlier work (Sood et al.,
2000) it was shown that nitrate reductase activity was inhibited by KN treatment and
thus, the decreased assimilation of NO– to NH+ may inhibit GS activity.
3
4
nmol NADH oxidized/h/cotyledon
nmol NADH oxidized/h/cotyledon
Concentration, mM
Concentration, mM
Fig. 5. Changes in glutamate synthase activity
Fig. 6. Changes in glutamate synthase activity
with different ambient concentrations of KNO3
with different ambient concentrations of KNO3
(X—X), NH Cl (
NO (o—o) in
4
•—•) and NH4
3
(X—X), NH Cl (
NO (o—o) in
4
•—•) and NH4
3
the cotyledons of (a) dark grown and (b) light
the cotyledons of (a) dark grown and (b) light
grown dH O treated radish seedlings. Vertical bars
2
grown KN treated radish seedlings. Vertical bars
represent ±SD (wherever bars are absent, SD is
represent ±SD (wherever bars are absent, SD is
so small that it is within the symbol).
so small that it is within the symbol).

Effect of different nitrogen sources and plant growth regulators on glutamine . . .
53
Glutamate synthase, is the second enzyme of “glutamate synthase cycle”. Although,
in higher plants, Fd and NADH dependent GOGAT have been reported, it is suggested
that NADH-GOGAT may be more important in N-metabolism during the earlier growth
period and in dark. In the present study, considerable NADH-GOGAT activity was
recorded in the absence of N in the medium (Fig. 5). Supplementation with NH+ or
4
NO– alone did not affect the activity to a larger extent. However, addition of NH NO
3
4
3
in dH O-control seedlings promoted NADH-GOGAT in dark (Fig. 5a). In contrast
2
to this, KN-treated seedlings, in dark, recorded higher NADH-GOGAT activity when
NH Cl was the N-source, while KNO and NH NO had slightly inhibitory effects
4
3
4
3
(Fig. 6a). No considerable effect of N-source was evident in dark-grown GA-treated
seedlings (Fig. 7a), while in ABA-treated seedlings, all N-sources promoted the ac-
tivity at low concentrations (Fig. 8a).
nmol NADH oxidized/h/cotyledon
nmol NADH oxidized/h/cotyledon
Concentration, mM
Concentration, mM
Fig. 7. Changes in glutamate synthase activity
Fig. 8. Changes in glutamate synthase activity
with different ambient concentrations of KNO
with different ambient concentrations of KNO
3
3
(X—X), NH Cl (•—•) and NH NO (o—o) in the
(X—X), NH Cl (
NO (o—o) in the
4
4
3
4
•—•) and NH4
3
cotyledons of (a) dark grown and (b) light grown
cotyledons of (a) dark grown and (b) light grown
GA treated radish seedlings. Vertical bars repre-
ABA treated radish seedlings. Vertical bars re-
sent ±SD (wherever bars are absent, SD is so
present ±SD (wherever bars are absent, SD is so
small that it is within the symbol).
small that it is within the symbol).

54
Ch. R. Sood et al.
In light-grown seedlings, NADH- GOGAT activity was inhibited when compared
to dark-grown seedlings. In dH O-control seedlings, both KNO and NH Cl promoted
2
3
4
NADH-GOGAT activity while NH NO showed inhibitory effects (Fig. 5b). In KN
4
3
treatment, addition of NH NO inhibited NADH-GOGAT to some extent while in the
4
3
other two N-sources no clear trend was discernible (Fig. 6b). As in dark grown seed-
lings, the effect of all the three N-sources was unclear in light grown GA treated seed-
lings (Fig. 7b). The activity of NADH-GOGAT was promoted by both NH NO and
4
3
NH Cl in ABA-treated seedlings while, KNO was effective only at higher concentra-
4
3
tions (Fig. 8b). From the above mentioned changes in NADH-GOGAT activity no clear
conclusions can be drawn. This may, perhaps, be due to the fact Fd-GOGAT activity
was not studied and it is desirable that changes in Fd-GOGAT are also monitored.
However, it appears that addition of NH+ promoted NADH-GOGAT activity to some
4
extent. Earlier work has also shown that NH+ can induce the expression of NADH-
4
GOGAT in maize seedlings (Handa et al., 1985), in the roots of alfalfa (Groat and
Vance, 1982) and in the cotyledons of Phaseolus vulgaris (Leon et al.,1990).
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