Biologia 70/4: 478485, 2015

Section Botany
DOI: 10.1515/biolog-2015-0060

Response of nitrogen assimilating enzymes during in vitro culture

of Argyrolobium roseum
Darima Habib1, Muhammad Zia2*, Yamin Bibi3, Bilal Haider Abbasi2
& Muhammad Fayyaz Chaudhary4
1

University of Haripur Khayber Pakhtunkhwa Pakistan

Department of Biotechnology, Quaid-i-Azam University, Islamabad, 45320 Pakistan
3
Department of Botany, PMAS University of Arid Agriculture, Rawalpindi, 44000 Pakistan
4
PINSAT, Preston University Islamabad Pakistan; e-mail: ziachaudhary@gmail.com
2

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Abstract: Nitrogen assimilating enzymes play curtail role during un-dierentiation and re-dierentiation of plant cells. To
investigate role and pattern of glutamine synthetase (GS), nitrate reductase (NR) and glutamate dehydrogenase (GDH)
during in vitro life cycle of Argyrolobium roseum this study was conducted. The concentrations of these enzymes were
determined during seed germination; callus induction from leaf, stem and root explants; shoot regeneration from callus; root
development and acclimatization stages. GS and NR enzymes showed ascending pattern during in vitro plant development
from seed while GDH concentration decreased during this process. Completely reverse pattern was showed by these enzymes
during callogenesis and proliferation phase. Increase in GS and NR activities was noticed in regenerated leaves and stem
during shoots and roots developmental phases; and vice verse for GDH. The acclimatization stress also up lifted NR and
GS activities in leaf, stem and root tissues. This study highlights the importance of nitrogen assimilating enzymes (NR,
GS, and GDH) during growth and development of A. roseum in vitro culture.

Introduction

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Key words: Argyrolobium roseum; glutamate dehydrogenase; glutamine synthetase; nitrogen assimilating enzymes; nitrate
reductase.

Switching the cells to undierentiated mass from organized cells and restoration again towards dierentiation from callus is controlled at the gene level, however,
indirectly is the change in enzymatic pattern. Carbon
and nitrogen are key elements required for synthesis of
biomolecules for biochemical functioning and building
of cellular components. The photosynthetic organisms
have the ability to x carbon from CO2 to biomolecules;
on the other hand, they mostly depend upon inorganic
form of nitrogen such as nitrate, nitrite and ammonia
to fulll the requirement.
For nitrogen assimilation, a number of key factors
are involved responsible for conversion of available nitrogen to compatible nitrogen. The most available form
of nitrogen, nitrate, rst reduces to nitrite by nitrate
reductase (NR) present in the cytosol (Meyer & Stitt
2001). This enzyme is a homodimer, each monomer being associated with three prosthetic groups: avin adenine dinucleotide, a haem, and a molybdenum cofactor. The activity of NR is coordinated with the rate of
photosynthesis and the availability of carbon skeleton
by both transcriptional and post translational controls
(Huber et al. 1994). Activity of NR occurs in both roots
* Corresponding author

c 2015 Institute of Botany, Slovak Academy of Sciences

and shoots but is spatially separated between the cytoplasm. After nitrate reduction, nitrite is translocated
to the chloroplast where it is reduced to ammonium
by the second enzyme of the pathway; nitrite reductase
(NiR). Ammonium, originating from nitrate and nitrite
reduction, and from amino acid recycling, assimilates
in the plastid/chloroplast by GS/GOGAT cycle (Lea
& Forde 1994). The glutamine synthetase (GS) is responsible to x ammonium to glutamine. Glutamine
subsequently reacts with 2-oxoglutarate to form two
molecules of glutamate and this step is catalyzed by
the glutamate synthase (GOGAT). In addition to the
GS/GOGAT cycle, three other enzymes also participate in ammonium assimilation that are asparagin synthetase (AS); carbamoylphosphate synthase (CPSase);
and glutamate dehydrogenase (GDH). AS generates
glutamate and asparagine by transferring amido group
of glutamine to aspartate (Lam et al. 1996). While in
plastids, CPSase forms carbamoylphosphate using bicarbonate, ATP and ammonium or the amide group of
glutamine. Carbamoylphospahte works as precursor for
citrulline and arginine. Alternatively under high level
of ammonium stress NADH-GDH in mitochondria also
incorporate ammonium into glutamate (Skopelitis et al.
2006). However, the major catalytic activity for GDH in

N-assimilating enzymatic pattern during A. roseum culture

Material and methods

was used for enzyme assays. Fresh tissues (300 mg) were incubated in screw-capped test tubes containing 12 mL potassium phosphate buer (pH 7.5), 0.6 mL of 25% proponal and
0.9 mL distilled water. Tubes were airtight and nitrogen gas
was bubbled through each tube for 2 min and transferred to
shaking water bath set at 30 C. After 1 min, 1.5 mL KNO3
was added and incubated for 1 hr. The NO2 produced by
the action of NR enzyme was determined by drawing 0.5 mL
aliquot of the incubation medium and added to the tubes
containing 0.5 mL distilled water, 1.0 mL sulfonamide and
1 mL NED (0.01%) solution. Tubes were thoroughly shaken
and allowed standing for 15 min. The O.D was recorded at
540 nm and enzyme activity was expressed as mol NO2 g
fresh wt1 h1 at 25 2 C.
In order to calculate the amount of NO2 contained in
the sample, a standard curve was prepared in the same way
as sample but using aliquots of 0.5 mL of NaNO2 (containing
0140 mol mL1 NO2 ).

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Determination of GS activity
The enzyme activity was determined according to the
method described by Rowe et al. (1970). Plant material (1 g)
was ground in pre-chilled pestle and mortar with extraction
medium comprised of 100 mM imidazole buer (pH 7.5),
DTT (2.0 M) and MgCl2 (100 mM) resulting crude enzyme
formation.
In a test tube, 0.2 mL crude enzyme preparation was
added to the pre incubated reaction medium, which consisted of imidazole buer (33 M; pH 7.5), Naglutamate
(50 M) pH 7, ATP (10 M), DTT (0.1 M), MgCl2
(20 M) and freshly prepared hydroxylamine (100 M; pH
7.0). The test tubes were placed in a shaking water bath at
37 C and after 30 min, 1.5 mL of FeCl3 solution (0.37 M
FeCl3 , 0.67 N HCl, 0.2 N TCA) was added and tubes were
transferred to the beaker containing ice for 15 min. Later on
the tubes were centrifuged at 3000 g for 30 min at 4 C. Supernatant was taken and O.D was recorded at 535 nm. Enzyme activity was expressed as mol L-glutamic acid monohydroxamate mg protein1 min1 at 252 C.

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plant cells has been reported to be glutamate deamination (Masclaux-Daubresse et al. 2006; Purnell & Botella
2007).
There are many reports on concentration of these
enzymes at dierent stages as seedling, callus, regenerated shoots and roots (Jaworski 1971; Magalhaes & Huber 1991; Robinson et al. 1992; Kormutak & Vookova
1997; Grabowska et al. 2011) and processes as callogenesis, organogenesis and acclimatization (Hardy &
Thorpe 1990; Li & Oaks 1993; Vazquez et al. 1994; Brewitz et al. 1996; Chanda et al. 1998; Arslan & G
ulery
uz
2005; Fontaine et al. 2006; Fontaine et al. 2012). However, no information is available on the development of
nitrogen assimilating enzymes during in vitro life cycle
of plants.
The present study was conducted to examine the
complete pattern of nitrogen assimilating enzymes (nitrate reductase, glutamate dehydrogenase, glutamine
synthetase) during in vitro life cycle of Argyrolobium
roseum and to nd the role of these enzymes during
de-dierentiation and re-dierentiation. In this study,
we demonstrate the estimation of nitrogen assimilating enzymes during in vitro-grown plants from seeds;
calli induced from dierent explants, callus proliferation, shoot and root development, and in dierent parts
of the acclimatized plant.

479

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Culture conditions and plant material

Nitrogen assimilating enzymes concentrations were determined during the in vitro germination of seeds, callogenesis,
organogenesis, and acclimatization stages of Argyrolobium
roseum. The methodology for in vitro culturing of A. roseum was followed as described by Habib et al. (2014). In
short, Nitrate Reductase (NR); Glutamine synthetase (GS);
and Glutamate dehydrogenase (GDH) enzymes were studied in leaf, stem, and roots parts of in vitro-grown plants
from the rst week of germination till the 8th week. The
same enzymes were also studied during callus development
from leaf, hypocotyls, and root explants on MS (Murashige
& Skoog 1962) medium containing plant growth regulators (1.0 mg L1 NAA and 0.5 mg L1 BA) and additives
(AgNO3 and PVP 0.5 mg L1 each) from 2nd week of callus
initiation till the 18th week. Furthermore, during shoot induction on MS medium supplemented with 2.0 mg L1 BA
and 1.5 mg L1 NAA with AgNO3 and PVP (0.5 mg L1
each), both leaf and stem explants were processed for enzymes assays starting from the 1st week of shoot initiation
till the 10th week. The leaf, stem, and root explants of plants
rooted on 0.5 mg L1 IBA were subjected to enzyme assay
during root development stage. These three explants were
also subjected to study enzymes concentrations during acclimatization stage. Total soluble protein determination was
also performed following the method of Bradford (1976).
All the readings were drawn from Agilent 8453 UV-Visible
Spectrophotometer.
Determination of NR activity
NR activity was determined by the intact tissue method as
described by Jaworski (1971). A 25 mL screw capped test
tube with central rubber core and wrapped with black paper

Determination of GDH activity

The enzyme was assayed by the method of Kates & Jones
(1964). Sample material (1 g) was ground in pre chilled pestle and mortar with 3 mL extraction medium that consisted
50 mM Tris HCl buer (pH 8.0), MgCl2 (1.0 mM), DTT
(1.0 mM), MnCl2 (1.0 mM) and subjected to centrifugation at 10,000 g for 45 min at 4 C. Supernatant (0.1 mL)
was reacted with 3 mL reaction mixture in a test tube and
placed in water bath. Reaction mixture comprised of 33 M
Tris HCl buer (pH 9.0), 100 M NH4 Cl, 1.0 M CaCl2 ,
1.0 M MgCl2 , 0.1 M DTT, 0.3 M NADH, 4.0 M ketoglutarate (pH 7.0) was pre-warmed at 37 C for 20 min.
Cuvette was inserted in spectrophotometer chamber after
shaking it well and O.D was recorded at 340 nm. Enzyme
activity was expressed as mol NAD(P)H decreased mg
protein1 min1 at 25 2 C.
Experimentation and data analysis:
All the enzyme assays were performed in triplicate and values are presented as mean standard deviation. Percent
variation in enzyme concentration at each stage was calculated as:
Percent variation = ((nal concentration initial concentration)/initial concentration)*100
Data obtained were analyzed using one-way analysis
of variance (ANOVA) with repeated measures followed by

480

D. Habib et al.

Table 1. Nitrate reductase (NR; mol NO2 g fresh wt1 h1 ), glutamate dehydrogenase (GDH; mol NAD(P)H decreased mg
protein1 min1 ) and glutamine synthetase (GS; mol L-glutamic acid monohydroxamate mg protein1 min1 ) enzymes activities
during in vitro plant development of A. roseum. Results are expressed as mean SD of three replicates. Means sharing at least one
letter are not signicantly dierent determined by Tukey test at the 0.05 level.
NR

GDH

GS

Week
Leaf
1
2
3
4
5
6
7
8

41.1
42.0
44.4
47.0
47.1
50.0
50.1
53.9

Stem

1.3e
2.3de
2.9d
2.2c
1.5c
1.1b
2.9ab
1.8a

24.1
25.0
29.9
30.0
32.4
37.0
39.1
42.1

2.0d
2.5d
1.1c
1.4c
2.5c
2.1b
1.8ab
1.5a

Root
38.3
39.8
41.0
42.0
42.9
45.0
46.1
46.5

2.1b
1.2b
1.7b
2.4ab
1.7ab
2.5a
1.9a
1.9a

Leaf
40.1
39.1
37.0
34.1
33.1
31.0
28.7
28.0

2.9a
1.7a
3.0ab
2.4b
2.0b
1.3bc
2.9bc
1.6c

Stem
31.8
29.1
29.0
27.1
26.9
24.3
22.1
20.1

1.7a
2.7a
2.5a
1.5b
1.8b
1.1c
2.5cd
2.5d

Root
19.0
16.0
15.9
15.0
14.1
14.0
12.9
12.0

2.0a
3.0ab
1.4ab
1.6b
1.4b
1.6b
2.5c
1.6c

Leaf
18.8
19.6
20.0
23.0
24.5
26.1
29.8
32.2

3.0c
3.5c
2.8c
1.7bc
1.6b
1.7ab
3.7a
2.5a

Stem
6.8
8.0
9.7
10.3
14.9
18.0
20.1
22.9

1.4d
1.9c
2.8c
2.1c
2.0b
2.1ab
3.1a
1.5a

Root
6.0
8.9
11.0
11.4
12.9
13.0
14.1
15.4

2.0c
1.3c
1.7b
2.9b
2.1ab
2.0ab
2.7a
1.5a

Table 2. Nitrate reductase (NR; mol NO2 g fresh wt1 h1 ), glutamate dehydrogenase (GDH; mol NAD(P)H decreased mg
protein1 min1 ) and glutamine synthetase (GS; mol L-glutamic acid monohydroxamate mg protein1 min1 ) enzymes activities
during callogenesis from leaf, stem and root explants of A. roseum. Results are expressed as mean SD of three replicates. Means
sharing at least one letter are not signicantly dierent determined by Tukey test at the 0.05 level.
GDH

NR
Week

43.1
43.1
42.1
41.7
40.0
40.0
39.2
39.0
38.1
37.9
37.1
36.9
36.4
36.1
36.0
35.5
34.0

3.1a
2.1a
2.8a
1.3ab
1.8b
2.9b
1.6b
1.7b
2.4b
3.0b
3.2b
2.0bc
2.4bc
1.5c
2.9c
2.2c
2.8c

48.9
48.6
48.1
47.9
47.3
47.0
46.4
46.9
46.0
44.9
44.1
44.0
42.5
42.1
39.9
37.3
35.1

4.0a
2.6a
3.0a
1.6a
2.4a
2.3a
2.0ab
3.0ab
2.2ab
2.8b
3.2b
2.3b
3.0b
2.3b
1.6b
2.4c
2.7c

Leaf
39.6
43.0
44.9
46.9
48.0
48.0
49.4
50.0
52.2
54.3
56.8
59.0
60.0
62.3
64.3
66.3
66.0

3.0e
2.9d
2.0c
1.4c
2.6dc
1.9dc
2.8dc
1.5dc
2.8d
1.7d
3.0c
2.0bc
3.1bc
2.6b
1.9b
1.9a
2.9a

Stem

29.9
37.0
39.1
41.9
43.2
44.1
46.7
48.8
49.0
50.0
51.8
53.0
53.0
54.2
55.4
58.7
59.8

comparisons with P-values 0.05 were considered signicantly dierent according to Tukey test.

Root

Leaf

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2.9a
3.1a
1.9a
2.8a
2.8ab
1.4b
2.5b
2.7b
2.7b
2.0b
2.5bc
2.0bc
2.9bc
2.2c
1.6c
3.1c
2.2c

Root

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1 55.1
2 54.8
3 54.1
4 54.0
5 51.1
6 50.9
7 50.5
8 50.1
9 50.1
10 49.9
11 48.3
12 43.0
13 43.0
14 40.1
15 38.9
16 38.6*
17 37.8

Stem

2.0h
2.2g
2.1f
3.0f
1.7e
3.1e
2.5d
2.5c
2.1c
3.0c
2.0bc
1.1b
2.5b
3.0ab
2.5ab
1.9a
1.3a

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Leaf

14.0
19.1
22.0
23.0
30.0
32.7
34.6
40.0
41.0
43.9
46.2
48.0
49.9
52.2
53.0
53.9
54.9

2.2f
2.9ef
2.4e
1.4e
2.0d
3.0d
2.4d
2.5cd
2.3cd
2.1c
1.1bc
3.0b
2.0b
3.0ab
2.3a
1.9a
2.3a

33.1
32.9
32.6
31.7
31.0
31.0
30.0
30.7
28.1
28.0
26.9
24.3
20.3
19.6
18.9
16.0
14.4

2.8a
2.5a
1.3a
1.6ab
2.9ab
1.7ab
2.0b
1.1b
2.0bc
1.0bc
1.6c
1.6d
2.7e
2.1e
1.8e
1.7ef
1.8g

GS
Stem
22.0
22.7
20.7
20.0
19.0
18.1
17.9
16.8
15.3
15.0
14.0
13.9
13.6
13.1
12.9
12.9
12.1

2.9a
1.4a
2.6ab
1.9b
2.7b
2.0bc
2.9bc
2.1c
2.9c
2.1c
2.0cd
2.5cd
1.6cd
1.8d
2.0d
1.8d
2.5d

Root
16.3
17.6
17.1
16.0
15.9
14.0
14.2
14.0
13.1
12.3
12.0
11.1
10.1
10.2
9.9
9.3
9.1

3.0ab
1.4a
2.4a
2.3ab
1.7ab
1.5b
1.9b
2.0b
1.5bc
2.4c
1.3c
2.7c
1.8d
2.8d
1.6d
1.1d
1.2d

Results

enzyme activity in in vitro plant developed on plain MS

medium. In vitro grown leaves exhibited 71.7% increase
in GS activity while stem and roots resulted 238% and
157% increase, respectively (Table 1).

Nitrogen metabolizing enzyme pattern during in vitro

seedling
Nitrate reductase (NR), Glutamate dehydrogenase
(GDH) and Glutamine synthetase (GS) enzyme activities were determined in leaves, stem and root parts of
in vitro-grown Argyrolobium roseum plants. Increased
pattern in the activity of NR was observed from the 1st
week until the last week of in vitro developed plants. Activity of NR raised upto 74.81% and 31.14% in in vitro
grown stem and leaves, respectively whereas, roots presented 21.3% increase (Table 1). GDH showed decrease
pattern during in vitro development of plants. In vitro
grown leaves showed 30.21% decrease in enzyme activity, while stem and roots showed 36.56% and 37.7%
decrease in pattern, respectively from the 1st week until 8th week. GS demonstrated an increasing pattern of

Nitrogen assimilating enzymes pattern during callus

formation and proliferation
Explants of eight weeks old in vitro seedlings were cut
into small pieces and transferred to callus induction
medium. Enzyme activities of NR, GDH and GS were
studied from 2nd week of callus formation to the 18th
week. NR presented decrease in pattern as calli developed from root, stem and leaf explants. Leaf, stem and
root originated calli showed 31, 21 and 28% decrease
in NR activity, respectively observed from the 2nd to
the 18th week of callus formation and proliferation (Table 2). Similarly, GS also showed decline pattern during
callogenesis. Leaf originated calli reported 56% decrease
in GS enzyme level as compared to stem (45%) and
roots (44%). GDH presented completely reverse pattern in enzyme activity as compared to GS and NR.

N-assimilating enzymatic pattern during A. roseum culture

481

Table 3. Nitrate reductase (NR; mol NO2 g fresh wt1 h1 ), glutamate dehydrogenase (GDH; mol NAD(P)H decreased mg
protein1 min1 ) and glutamine synthetase (GS; mol L-glutamic acid monohydroxamate/ mg protein1 min1 ) enzymes activities
during in vitro shoot induction of A. roseum. Results are expressed as mean SD of three replicates. Means sharing at least one letter
are not signicantly dierent determined by Tukey test at the 0.05 level.
NR

GDH

GS

Week
Leaf
1
2
3
4
5
6
7
8
9
10

30.0
31.1
32.2
33.4
34.0
34.6
35.0
36.1
38.4
39.1

Stem

2.9b
1.9b
2.7b
2.0b
2.8b
2.6ab
2.9ab
2.0ab
3.1a
2.1a

22.7
23.0
23.2
23.7
24.0
24.6
25.1
26.5
28.7
29.3

Leaf

1.9d
2.9c
2.2bc
1.6bc
3.2b
2.1b
1.9b
1.7ab
2.4a
2.0a

61.2
61.0
58.4
57.1
56.1
55.7
55.0
54.3
54.1
53.2

Stem

3.0a
1.6a
2.9ab
2.2b
1.8b
3.0bc
3.1c
2.0c
2.7c
3.0c

55.3
55.0
54.2
53.5
52.8
52.0
51.1
49.9
49.2
48.1

Leaf

2.4a
2.3a
1.0ab
2.0b
2.4b
3.0b
2.4bc
2.1bc
1.4bc
2.4b

11.1
11.4
12.4
13.0
13.2
14.0
14.4
15.1
16.0
16.2

Stem

1.1c
2.3c
1.8bc
1.5b
1.4b
1.9ab
2.6ab
1.6ab
1.8a
2.3a

8.8
9.0
9.1
9.6
10.0
10.0
10.2
10.9
11.3
12.0

2.6bc
1.7bc
1.9bc
2.7b
1.2b
2.0b
1.8b
1.8b
2.3ab
2.3a

GDH

Week

44.3
46.3
47.5
49.0
49.5
50.8
52.9
53.6
55.9
56.0

2.9d
1.9c
2.7c
2.3bc
3.0bc
3.2bc
2.1b
1.9b
1.8a
2.8a

34.1
35.0
35.0
37.0
38.0
39.0
39.1
39.9
40.1
41.1

1.9c
2.8bc
2.0bc
1.5b
2.2b
2.1ab
2.0ab
3.1ab
2.1ab
2.0a

Root
38.1
38.3
39.4
40.0
41.3
42.0
42.0
42.3
42.9
43.6

2.7b
1.8b
2.5b
2.0ab
2.6ab
2.7a
3.0a
2.1a
2.8a
1.9a

Leaf
51.0
50.7
50.0
48.1
47.8
47.1
46.2
45.1
44.9
44.2

3.0a
2.6a
1.8a
3.0a
2.8a
1.9ab
2.6ab
2.9b
2.0b
1.0b

Stem

46.2
46.0
45.2
44.1
44.0
42.0
41.2
40.0
38.8
38.3

Au
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1
2
3
4
5
6
7
8
9
10

Stem

2.1a
2.0a
2.9a
1.8ab
2.5ab
2.3b
2.7b
3.1b
2.0b
2.9b

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Leaf

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NR

Table 4. Nitrate reductase (NR; mol NO2 g fresh wt1 h1 ), glutamate dehydrogenase (GDH; mol NAD(P)H decreased mg
protein1 min1 ) and glutamine synthetase (GS; mol L-glutamic acid monohydroxamate mg protein1 min1 ) enzymes activities
during in vitro root organogenesis of A. roseum

Root

40.0
39.5
38.1
37.7
36.6
34.2
33.1
33.0
32.9
32.0

3.0a
2.1a
3.0a
2.6ab
3.1ab
2.3b
2.4b
2.0b
2.4b
2.4b

Leaf

18.2
19.1
20.0
23.1
25.0
25.1
26.7
27.4
28.7
30.2

2.6c
2.2c
1.0c
1.1bc
2.1b
1.7b
1.2ab
2.4ab
2.0a
1.5a

GS
Stem
13.1
14.0
14.0
14.0
15.0
15.6
16.0
16.1
16.4
16.9

1.6b
2.7b
1.5b
1.7b
1.1ab
2.0ab
2.7a
1.8a
1.5a
1.3a

Root
10.4
11.9
12.2
13.0
14.0
14.0
14.9
15.3
15.9
16.7

1.9b
2.6b
1.5b
2.8b
1.7ab
2.3ab
1.9ab
1.4ab
1.4ab
2.0a

Results are expressed as meanSD of three replicates. Means sharing at least one letter are not signicantly dierent determined by
Tukey test at the 0.05 level.

Increase in pattern was noticed in calli derived from

leaf, stem and root explants. Leaf derived calli showed
66% increase in enzyme activity, whereas stem and root
derived calli exhibited 100% and 292%, respectively, increase during callus formation and proliferation (Table 2).

Nitrogen metabolizing enzyme activities in regenerate

leaves and stem
Shoot originated on MS medium were subjected for the
analysis of nitrogen metabolizing enzymes from the rst
week to the 10th week. NR increased almost equally
both in stem and leaf i.e. 30% and little bit more increase was observed in GS; 46% in leaves and 36% in
stem (Table 3). Decrease (13%) in GDH was observed
in both leaf and stem parts during shoot organogenesis.
Activities of nitrogen assimilating enzymes during root
development and acclimatization:
During root induction and acclimatization, NR and GS
increased while GDH showed decrease in concentration.
During root initiation, more increase in NR (26%) was
observed in leaves (Table 4) as compared with root and
stem however, increase in NR activity pattern changed

during acclimatization and high increase in root was

observed (Table 5). Increase in GS was also observed in
all three parts of A. roseum during root induction and
acclimatization. During root induction GS activity was
observed higher in leaves (66%) following root (59%)
and stem (29%) (Table 4). However, high activity was
observed stem part following leaf and root, respectively.
Roots showed 20% decrease in GDH enzyme level during root induction and this decrease was also observed
in leaf and stem parts (Table 4). The same pattern of
decrease in GDH was noticed during acclimatization
process (Table 5).
Discussion
Pattern of nitrate reductase (NR) during A. roseum life
cycle
NR is a light inducible enzyme and its activity relates with both photosynthesis and morphogenesis. NR
showed increase in activity as leaf > stem > root parts
of in vitro grown A. roseum from seeds. The increase
was pronounced in photosynthetic parts as compared
with non-photosynthetic (Fig. 1). It has been postulated that light acting through photosynthesis promotes

482

D. Habib et al.

Table 5. Nitrate reductase (NR; mol NO2 g fresh wt1 h1 ), glutamate dehydrogenase (GDH; mol NAD(P)H decreased mg
protein1 min1 ) and glutamine synthetase (GS; mol L-glutamic acid monohydroxamate mg protein1 min1 ) enzymes activities
during acclimatization of in vitro developed plants of A. roseum. Results are expressed as mean SD of three replicates. Means
sharing at least one letter are not signicantly dierent determined by Tukey test at the 0.05 level.
NR

GDH

GS

Week
Leaf
57.1
58.0
61.8
62.9
63.4
64.1
66.4
67.9

2.5c
2.1c
1.4bc
3.0bc
2.3b
1.9b
2.7a
1.4a

42.1
43.0
43.4
44.0
46.9
47.1
48.0
48.8

2.1c
1.7b
3.0b
1.4b
2.9ab
1.1ab
1.9a
3.0a

Root
44.1
44.5
45.0
45.1
46.9
48.9
52.3
55.0

2.5e
3.2e
2.2e
3.0e
1.9d
2.3c
2.8b
2.0a

Leaf
43.2
42.0
41.2
40.9
40.2
40.0
39.2
39.0

2.1a
1.7ab
3.0b
1.4b
2.9b
1.1b
1.9b
3.0b

Stem
36.6
36.0
35.1
34.2
34.0
32.0
31.0
30.0

1.3a
3.0a
2.1ab
1.0b
1.4b
3.0c
1.5cd
1.6d

Root
30.0
27.9
27.0
26.3
25.3
24.1
22.1
20.1

3.0a
1.0b
2.8b
1.9bc
2.8bc
3.1bc
1.9c
1.3c

Leaf
31.6
32.0
32.0
33.0
35.0
36.0
37.1
38.0

2.4d
1.7cd
2.0cd
2.8c
2.1b
1.5b
2.9ab
1.6a

Stem
16.1
17.4
19.0
20.2
20.9
22.0
22.9
24.0

2.8c
1.9c
2.8bc
1.5b
1.9b
1.7ab
2.7ab
1.9a

Root
16.0
16.9
17.1
17.9
18.0
18.9
19.1
19.1

2.7b
2.0ab
2.8ab
1.4ab
1.9ab
3.0a
2.0a
1.7a

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1
2
3
4
5
6
7
8

Stem

Fig. 1. Complete pattern of nitrogen assimilating enzymes; (NR), nitrate reductase; (GDH), glutamate dehydrogenase; (GS), glutamine
synthetase during in vitro life cycle of A. roseum. (The gaps between the lines are created to separate dierent phases).

synthesis of carbohydrates; producing NADH necessary

for NR activity (Li & Oaks 1993; Arslan & G
ulery
uz
2005). Present study provides the evidence that NR activity amplies while plant start photosynthesis and increase in biomass too.
When leaf, stem and root explants inoculated on
callus initiation medium, decline in NR activity was
observed. However, leaf explants derived calli showed
zigzag pattern as compared to others (Fig. 1) might
be due to explants; origin of calli source, the greener
and photosynthetic in nature. Leaf is the main site
for nitrate reduction and such pattern was also noticed in corn leaf derived calli from 1st week to 10th
week (Remmler & Campbell 1986). The NR activity
decreased at higher rate at the end of calli proliferation stage might be due to depletion of nitrate from the
culturing medium and also decrease in photosynthetic
activity, the calli cells started to die.
High NR activity was observed in leaves as compared to stem during the regeneration phase (Fig. 1).
It has been reported that photosynthetic electron ow

from PSI (photo system) and PSII are signicantly important in controlling the expression of NR enzyme and
there is a positive correlation between growth, protein
content and NR activity (Dwivedi et al. 1984). Continuous increase in NR pattern has also been reported
during regeneration period of tobacco from calli (Hardy
& Thorpe 1990). The regeneration phase requires more
energy for cellular functioning, increase in biomass and
also switching towards development demands some extra liabilities. This situation continued during root development, however, increase was more pronounced as
compared with shoot development. The boost might be
due to two possibilities; one refreshing the culture media and second emergence of root responsible for uptake of nitrate and other nutrients. Lillo & Appenroth
(2001) have reported that growth phenomenon increase
the level of nitrogen assimilating enzymes in plants.
At the acclimatization stage; NR activity was maximum in leaf, stem and roots as compared with other
stages. This might be due to that, plants are fully developed; photosynthetic activity has been started while no

N-assimilating enzymatic pattern during A. roseum culture

Pattern of glutamine synthetase (GS) in A. roseum

One of the most important enzymes in nitrogen xation and ammonia incorporation into protein is GS
(Magalhaes & Huber 1991). Leaf showed high level of
GS activity as compared to stem and roots during in
vitro germination of A. roseum. Such increase in activity has also been reported in other plants (Mack &
Schjoerring 2002). This might be due to that plants assimilate most of their nitrogen in the leaves and during
the growth period and for normal growth, activities of
biosynthetic enzymes increase (Kiyomiya et al. 2001;
Tabuchi et al. 2005).
Activity of GS decreased as explants were placed
on callus induction medium (Fig. 1). Low activity of
GS in cells grown on NH4 containing medium has been
reported by several authors (Robinson et al. 1992; Florencio & Ramos 1985). Ammonium and nitrate uptakes
and their metabolism in cells signicantly determine the
biochemical characteristics of the callus. Leaf derived
calli reported fast decline as compared to the stem and
root. This might be due to the fact that source of calli
formation plays an important role in the biochemical
and physiological characteristics of calli. At the end of
proliferation phase, no drastic dierence in the activity of GS was observed in leaf, stem and root derived
calli. Calli absorb nitrate from the medium which result excess of ammonia at the lateral stage. It has been
reported that excess of ammonia inhibits GS activity
(Kormutak & Vookova 1997). Furthermore, decreased
pattern of GS might also be due to the medium which
is exhausted by various nutrients especially sucrose and
presence of phytohormones play synergistic role controlling the enzymatic activities.
Reverse pattern was observed by GS enzyme during regeneration phase as compared to callogenesis.
This increase in pattern continued even during root
development (Fig. 1). The activity of biosynthetic enzymes increases during the regeneration phase and
plant growth regulators enforce the callus to regenerate shoot; might be involved in regulation of certain

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Pattern of glutamate dehydrogenase (GDH) in A. roseum

During the growth process from seedling to plant; decrease in GDH activity was observed in all plant parts.
Normally, catabolic enzymes concentrations remain low
during growth and developmental processes. Decline in
ammonia and sucrose concentration with time in the
culture medium might also be responsible for low GDH
level in A. roseum. NAD(P)H GDH enzyme is more
responsive to stress conditions as compared to NADH
GDH and is localized in chloroplast of photosynthesizing tissues (Lam et al. 1996). Miyashita & Good (2008)
also suggested that under carbon deciency, NAD(H)GDH involves in the breakdown of amino acids and
the supply of respiratory substrate to the tricarboxylic
acid cycle because GDH do not require ATP for its
functioning while parallel enzyme, GS-GOGAT is ATPdependent (Fischer & Klein 1988).
GDH started to increase as calli started to develop
from explants and the raise continued during proliferation stage. At the end of proliferation phase, leaf initiated calli showed only 66% increase from starting stage
as compared to stem (100%) and root (292%). Many
fold increase in GDH activity in root explants derived
calli might be due to source and biochemical characteristics of explants. Excess of ammonia (20.1 mM) in
the medium also enhances GDH activity used to incorporate ammonium into amino acids (Kormutak &
Vookova 1997). Excess ammonia triggers the -subunit
of GDH, which involves in the synthesis of proteins and
increased activity of GDH may also be due to de novo
protein synthesis (Loulakakis & Roubelakis-Angelakis
1991). GDH also plays an important role in NH4 assimilation and alleviating the toxicity of excessive NH4
from tissue (Afzal et al. 2006). Callus culture contained
almost the double of GDH enzyme activity as compared
to explants initiated for calli origination. This determines a radical change in overall metabolic behavior of
callus. GDH enzyme is mainly localized in the phloem
cells (Dubois et al. 2003; Terc-Laforgue et al. 2004;
Fontaine et al. 2006; Fontaine et al. 2012), but its synthesis enhance in the mitochondria, when the ammonium concentration increase above a certain threshold
(Terc-Laforgue et al. 2004).
During shoot regeneration from calli, GDH activity
continued to decrease and this reduction preceded even
during root development and acclimatization stages.
The catabolic enzymes normally tend to decrease during the developmental process. Regeneration of shoots
and roots are organ development and requires amino
acids and protein at higher level, decreasing the activ-

ity of GDH. The expression of NAD(H)- or NADP(H)dependent GDH activity has an impact on amino acid
metabolism and in particular metabolism derived from
glutamate and glutamine, which could greatly inuence the translocation and use of amino acids that
can act as nitrogen transport molecules during plant
growth and development (Lea & Azevedo 2007; Lea et
al. 2007). This nutritional metabolic shift up during
callogenesis and shift down for the period of organogenesis inuenced by the nutrients and phytohormones
in the medium might also be responsible for decrease in
GDH (Soulen & Olsan 1969). According to Loulakakis
& Roubelakis-Angelakis (1991) excess ammonia is also
responsible for the increased activity of NADH-GDH in
leaves, shoots and roots. Glutamate is a primary product of ammonia assimilation in photosynthetic tissues,
although other amino acids such as alanine and aspartic
acid have been demonstrated to be important products
when nitrate is utilized as nitrogen source.

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exogenous carbohydrate is available and nitrate is available in the soil. Beside this, the plant has concluded the
developmental process and during growth period carbon and nitrogen skeleton are required at higher level.
A steady boost has been reported in tea plants during
acclimatization stage (Hajiboland et al. 2011). Lea et
al. (1990) accounted same observation while correlating
plant growth and nitrogen assimilation.

483

484

D. Habib et al.

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485

Received November 21, 2014

Accepted February 12, 2015