36 Response of nitrogen assimilating enzymes during in vitro culture of Argyrolobium roseum

Biologia 70/4: 478—485, 2015Section BotanyDOI: 10.1515/biolog-2015-0060

Response of nitrogen assimilating enzymes during in vitro cultureof Argyrolobium roseum

Darima Habib1, Muhammad Zia2*, Yamin Bibi3, Bilal Haider Abbasi2

& Muhammad Fayyaz Chaudhary4

1University of Haripur Khayber Pakhtunkhwa Pakistan2Department of Biotechnology, Quaid-i-Azam University, Islamabad, 45320 Pakistan3Department of Botany, PMAS University of Arid Agriculture, Rawalpindi, 44000 Pakistan4PINSAT, Preston University Islamabad Pakistan; e-mail: [email protected]

Abstract: Nitrogen assimilating enzymes play curtail role during un-differentiation and re-differentiation of plant cells. Toinvestigate 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 weredetermined during seed germination; callus induction from leaf, stem and root explants; shoot regeneration from callus; rootdevelopment and acclimatization stages. GS and NR enzymes showed ascending pattern during in vitro plant developmentfrom seed while GDH concentration decreased during this process. Completely reverse pattern was showed by these enzymesduring callogenesis and proliferation phase. Increase in GS and NR activities was noticed in regenerated leaves and stemduring shoots and roots developmental phases; and vice verse for GDH. The acclimatization stress also up lifted NR andGS 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.

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

Introduction

Switching the cells to undifferentiated mass from or-ganized cells and restoration again towards differentia-tion from callus is controlled at the gene level, however,indirectly is the change in enzymatic pattern. Carbonand nitrogen are key elements required for synthesis ofbiomolecules for biochemical functioning and buildingof cellular components. The photosynthetic organismshave the ability to fix carbon from CO2 to biomolecules;on the other hand, they mostly depend upon inorganicform of nitrogen such as nitrate, nitrite and ammoniato fulfill the requirement.For nitrogen assimilation, a number of key factors

are involved responsible for conversion of available ni-trogen to compatible nitrogen. The most available formof nitrogen, nitrate, first reduces to nitrite by nitratereductase (NR) present in the cytosol (Meyer & Stitt2001). This enzyme is a homodimer, each monomer be-ing associated with three prosthetic groups: flavin ade-nine dinucleotide, a haem, and a molybdenum cofac-tor. The activity of NR is coordinated with the rate ofphotosynthesis and the availability of carbon skeletonby both transcriptional and post translational controls(Huber et al. 1994). Activity of NR occurs in both roots

and shoots but is spatially separated between the cy-toplasm. After nitrate reduction, nitrite is translocatedto the chloroplast where it is reduced to ammoniumby the second enzyme of the pathway; nitrite reductase(NiR). Ammonium, originating from nitrate and nitritereduction, and from amino acid recycling, assimilatesin the plastid/chloroplast by GS/GOGAT cycle (Lea& Forde 1994). The glutamine synthetase (GS) is re-sponsible to fix ammonium to glutamine. Glutaminesubsequently reacts with 2-oxoglutarate to form twomolecules of glutamate and this step is catalyzed bythe glutamate synthase (GOGAT). In addition to theGS/GOGAT cycle, three other enzymes also partici-pate in ammonium assimilation that are asparagin syn-thetase (AS); carbamoylphosphate synthase (CPSase);and glutamate dehydrogenase (GDH). AS generatesglutamate and asparagine by transferring amido groupof glutamine to aspartate (Lam et al. 1996). While inplastids, CPSase forms carbamoylphosphate using bi-carbonate, ATP and ammonium or the amide group ofglutamine. Carbamoylphospahte works as precursor forcitrulline and arginine. Alternatively under high levelof ammonium stress NADH-GDH in mitochondria alsoincorporate ammonium into glutamate (Skopelitis et al.2006). However, the major catalytic activity for GDH in

* Corresponding author

c©2015 Institute of Botany, Slovak Academy of Sciences

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N-assimilating enzymatic pattern during A. roseum culture 479

plant cells has been reported to be glutamate deamina-tion (Masclaux-Daubresse et al. 2006; Purnell & Botella2007).There are many reports on concentration of these

enzymes at different stages as seedling, callus, regener-ated shoots and roots (Jaworski 1971; Magalhaes & Hu-ber 1991; Robinson et al. 1992; Kormutak & Vookova1997; Grabowska et al. 2011) and processes as callo-genesis, organogenesis and acclimatization (Hardy &Thorpe 1990; Li & Oaks 1993; Vazquez et al. 1994; Bre-witz et al. 1996; Chanda et al. 1998; Arslan & Guleryuz2005; Fontaine et al. 2006; Fontaine et al. 2012). How-ever, no information is available on the development ofnitrogen assimilating enzymes during in vitro life cycleof plants.The present study was conducted to examine the

complete pattern of nitrogen assimilating enzymes (ni-trate reductase, glutamate dehydrogenase, glutaminesynthetase) during in vitro life cycle of Argyrolobiumroseum and to find the role of these enzymes duringde-differentiation and re-differentiation. In this study,we demonstrate the estimation of nitrogen assimilat-ing enzymes during in vitro-grown plants from seeds;calli induced from different explants, callus prolifera-tion, shoot and root development, and in different partsof the acclimatized plant.

Material and methods

Culture conditions and plant materialNitrogen assimilating enzymes concentrations were deter-mined during the in vitro germination of seeds, callogenesis,organogenesis, and acclimatization stages of Argyrolobiumroseum. The methodology for in vitro culturing of A. ro-seum was followed as described by Habib et al. (2014). Inshort, Nitrate Reductase (NR); Glutamine synthetase (GS);and Glutamate dehydrogenase (GDH) enzymes were stud-ied in leaf, stem, and roots parts of in vitro-grown plantsfrom the first week of germination till the 8th week. Thesame enzymes were also studied during callus developmentfrom leaf, hypocotyls, and root explants on MS (Murashige& Skoog 1962) medium containing plant growth regula-tors (1.0 mg L−1 NAA and 0.5 mg L−1 BA) and additives(AgNO3 and PVP 0.5 mg L−1 each) from 2nd week of callusinitiation till the 18th week. Furthermore, during shoot in-duction on MS medium supplemented with 2.0 mg L−1 BAand 1.5 mg L−1 NAA with AgNO3 and PVP (0.5 mg L−1

each), both leaf and stem explants were processed for en-zymes assays starting from the 1st week of shoot initiationtill the 10th week. The leaf, stem, and root explants of plantsrooted on 0.5 mg L−1 IBA were subjected to enzyme assayduring root development stage. These three explants werealso subjected to study enzymes concentrations during ac-climatization stage. Total soluble protein determination wasalso performed following the method of Bradford (1976).All the readings were drawn from Agilent 8453 UV-VisibleSpectrophotometer.

Determination of NR activityNR activity was determined by the intact tissue method asdescribed by Jaworski (1971). A 25 mL screw capped testtube with central rubber core and wrapped with black paper

was used for enzyme assays. Fresh tissues (300 mg) were in-cubated in screw-capped test tubes containing 12 mL potas-sium phosphate buffer (pH 7.5), 0.6 mL of 25% proponal and0.9 mL distilled water. Tubes were airtight and nitrogen gaswas bubbled through each tube for 2 min and transferred toshaking water bath set at 30◦C. After 1 min, 1.5 mL KNO3was added and incubated for 1 hr. The NO2 produced bythe action of NR enzyme was determined by drawing 0.5 mLaliquot of the incubation medium and added to the tubescontaining 0.5 mL distilled water, 1.0 mL sulfonamide and1 mL NED (0.01%) solution. Tubes were thoroughly shakenand allowed standing for 15 min. The O.D was recorded at540 nm and enzyme activity was expressed as µmol NO2 gfresh wt−1 h−1 at 25 ± 2◦C.

In order to calculate the amount of NO2 contained inthe sample, a standard curve was prepared in the same wayas sample but using aliquots of 0.5 mL of NaNO2 (containing0–140 µmol mL−1 NO2).

Determination of GS activityThe enzyme activity was determined according to themethod described by Rowe et al. (1970). Plant material (1 g)was ground in pre-chilled pestle and mortar with extractionmedium comprised of 100 mM imidazole buffer (pH 7.5),DTT (2.0 M) and MgCl2 (100 mM) resulting crude enzymeformation.

In a test tube, 0.2 mL crude enzyme preparation wasadded to the pre incubated reaction medium, which con-sisted of imidazole buffer (33 µM; pH 7.5), Na–glutamate(50 µM) pH 7, ATP (10 µM), DTT (0.1 µM), MgCl2(20 µM) and freshly prepared hydroxylamine (100 µM; pH7.0). The test tubes were placed in a shaking water bath at37◦C and after 30 min, 1.5 mL of FeCl3 solution (0.37 MFeCl3, 0.67 N HCl, 0.2 N TCA) was added and tubes weretransferred to the beaker containing ice for 15 min. Later onthe tubes were centrifuged at 3000 g for 30 min at 4◦C. Su-pernatant was taken and O.D was recorded at 535 nm. En-zyme activity was expressed as µmol L-glutamic acid mono-hydroxamate mg protein−1 min−1 at 25±2◦C.

Determination of GDH activityThe enzyme was assayed by the method of Kates & Jones(1964). Sample material (1 g) was ground in pre chilled pes-tle and mortar with 3 mL extraction medium that consisted50 mM Tris HCl buffer (pH 8.0), MgCl2 (1.0 mM), DTT(1.0 mM), MnCl2 (1.0 mM) and subjected to centrifuga-tion 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 andplaced in water bath. Reaction mixture comprised of 33 µMTris HCl buffer (pH 9.0), 100 µM NH4Cl, 1.0 µM CaCl2 ,1.0 µM MgCl2, 0.1 µM DTT, 0.3 µM NADH, 4.0 µM α-ketoglutarate (pH 7.0) was pre-warmed at 37◦C for 20 min.Cuvette was inserted in spectrophotometer chamber aftershaking it well and O.D was recorded at 340 nm. Enzymeactivity was expressed as µmol NAD(P)H decreased mgprotein−1 min−1 at 25 ± 2◦C.

Experimentation and data analysis:All the enzyme assays were performed in triplicate and val-ues are presented as mean ± standard deviation. Percentvariation in enzyme concentration at each stage was calcu-lated as:Percent variation = ((final concentration – initial concen-tration)/initial concentration)*100

Data obtained were analyzed using one-way analysisof variance (ANOVA) with repeated measures followed by

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Table 1. Nitrate reductase (NR; µmol NO2 g fresh wt−1 h−1), glutamate dehydrogenase (GDH; µmol NAD(P)H decreased mgprotein−1 min−1) and glutamine synthetase (GS; µmol L-glutamic acid monohydroxamate mg protein−1 min−1) enzymes activitiesduring in vitro plant development of A. roseum. Results are expressed as mean ± SD of three replicates. Means sharing at least oneletter are not significantly different determined by Tukey test at the 0.05 level.

NR GDH GSWeek

Leaf Stem Root Leaf Stem Root Leaf Stem Root

1 41.1 ± 1.3e 24.1 ± 2.0d 38.3 ± 2.1b 40.1 ± 2.9a 31.8 ± 1.7a 19.0 ± 2.0a 18.8 ± 3.0c 6.8 ± 1.4d 6.0 ± 2.0c2 42.0 ± 2.3de 25.0 ± 2.5d 39.8 ± 1.2b 39.1 ± 1.7a 29.1 ± 2.7a 16.0 ± 3.0ab 19.6 ± 3.5c 8.0 ± 1.9c 8.9 ± 1.3c3 44.4 ± 2.9d 29.9 ± 1.1c 41.0 ± 1.7b 37.0 ± 3.0ab 29.0 ± 2.5a 15.9 ± 1.4ab 20.0 ± 2.8c 9.7 ± 2.8c 11.0 ± 1.7b4 47.0 ± 2.2c 30.0 ± 1.4c 42.0 ± 2.4ab 34.1 ± 2.4b 27.1 ± 1.5b 15.0 ± 1.6b 23.0 ± 1.7bc 10.3 ± 2.1c 11.4 ± 2.9b5 47.1 ± 1.5c 32.4 ± 2.5c 42.9 ± 1.7ab 33.1 ± 2.0b 26.9 ± 1.8b 14.1 ± 1.4b 24.5 ± 1.6b 14.9 ± 2.0b 12.9 ± 2.1ab

6 50.0 ± 1.1b 37.0 ± 2.1b 45.0 ± 2.5a 31.0 ± 1.3bc 24.3 ± 1.1c 14.0 ± 1.6b 26.1 ± 1.7ab 18.0 ± 2.1ab 13.0 ± 2.0ab

7 50.1 ± 2.9ab 39.1 ± 1.8ab 46.1 ± 1.9a 28.7 ± 2.9bc 22.1 ± 2.5cd 12.9 ± 2.5c 29.8 ± 3.7a 20.1 ± 3.1a 14.1 ± 2.7a8 53.9 ± 1.8a 42.1 ± 1.5a 46.5 ± 1.9a 28.0 ± 1.6c 20.1 ± 2.5d 12.0 ± 1.6c 32.2 ± 2.5a 22.9 ± 1.5a 15.4 ± 1.5a

Table 2. Nitrate reductase (NR; µmol NO2 g fresh wt−1 h−1), glutamate dehydrogenase (GDH; µmol NAD(P)H decreased mgprotein−1 min−1) and glutamine synthetase (GS; µmol L-glutamic acid monohydroxamate mg protein−1 min−1) enzymes activitiesduring callogenesis from leaf, stem and root explants of A. roseum. Results are expressed as mean ± SD of three replicates. Meanssharing at least one letter are not significantly different determined by Tukey test at the 0.05 level.

NR GDH GSWeek


1 55.1 ± 2.9a 43.1 ± 3.1a 48.9 ± 4.0a 39.6 ± 3.0e 29.9 ± 2.0h 14.0 ± 2.2f 33.1 ± 2.8a 22.0 ± 2.9a 16.3 ± 3.0ab

2 54.8 ± 3.1a 43.1 ± 2.1a 48.6 ± 2.6a 43.0 ± 2.9d 37.0 ± 2.2g 19.1 ± 2.9ef 32.9 ± 2.5a 22.7 ± 1.4a 17.6 ± 1.4a3 54.1 ± 1.9a 42.1 ± 2.8a 48.1 ± 3.0a 44.9 ± 2.0c 39.1 ± 2.1f 22.0 ± 2.4e 32.6 ± 1.3a 20.7 ± 2.6ab 17.1 ± 2.4a4 54.0 ± 2.8a 41.7 ± 1.3ab 47.9 ± 1.6a 46.9 ± 1.4c 41.9 ± 3.0f 23.0 ± 1.4e 31.7 ± 1.6ab 20.0 ± 1.9b 16.0 ± 2.3ab

5 51.1 ± 2.8ab 40.0 ± 1.8b 47.3 ± 2.4a 48.0 ± 2.6dc 43.2 ± 1.7e 30.0 ± 2.0d 31.0 ± 2.9ab 19.0 ± 2.7b 15.9 ± 1.7ab

6 50.9 ± 1.4b 40.0 ± 2.9b 47.0 ± 2.3a 48.0 ± 1.9dc 44.1 ± 3.1e 32.7 ± 3.0d 31.0 ± 1.7ab 18.1 ± 2.0bc 14.0 ± 1.5b7 50.5 ± 2.5b 39.2 ± 1.6b 46.4 ± 2.0ab 49.4 ± 2.8dc 46.7 ± 2.5d 34.6 ± 2.4d 30.0 ± 2.0b 17.9 ± 2.9bc 14.2 ± 1.9b8 50.1 ± 2.7b 39.0 ± 1.7b 46.9 ± 3.0ab 50.0 ± 1.5dc 48.8 ± 2.5c 40.0 ± 2.5cd 30.7 ± 1.1b 16.8 ± 2.1c 14.0 ± 2.0b9 50.1 ± 2.7b 38.1 ± 2.4b 46.0 ± 2.2ab 52.2 ± 2.8d 49.0 ± 2.1c 41.0 ± 2.3cd 28.1 ± 2.0bc 15.3 ± 2.9c 13.1 ± 1.5bc

10 49.9 ± 2.0b 37.9 ± 3.0b 44.9 ± 2.8b 54.3 ± 1.7d 50.0 ± 3.0c 43.9 ± 2.1c 28.0 ± 1.0bc 15.0 ± 2.1c 12.3 ± 2.4c11 48.3 ± 2.5bc 37.1 ± 3.2b 44.1 ± 3.2b 56.8 ± 3.0c 51.8 ± 2.0bc 46.2 ± 1.1bc 26.9 ± 1.6c 14.0 ± 2.0cd 12.0 ± 1.3c12 43.0 ± 2.0bc 36.9 ± 2.0bc 44.0 ± 2.3b 59.0 ± 2.0bc 53.0 ± 1.1b 48.0 ± 3.0b 24.3 ± 1.6d 13.9 ± 2.5cd 11.1 ± 2.7c13 43.0 ± 2.9bc 36.4 ± 2.4bc 42.5 ± 3.0b 60.0 ± 3.1bc 53.0 ± 2.5b 49.9 ± 2.0b 20.3 ± 2.7e 13.6 ± 1.6cd 10.1 ± 1.8d14 40.1 ± 2.2c 36.1 ± 1.5c 42.1 ± 2.3b 62.3 ± 2.6b 54.2 ± 3.0ab 52.2 ± 3.0ab 19.6 ± 2.1e 13.1 ± 1.8d 10.2 ± 2.8d15 38.9 ± 1.6c 36.0 ± 2.9c 39.9 ± 1.6b 64.3 ± 1.9b 55.4 ± 2.5ab 53.0 ± 2.3a 18.9 ± 1.8e 12.9 ± 2.0d 9.9 ± 1.6d16 38.6* ± 3.1c 35.5 ± 2.2c 37.3 ± 2.4c 66.3 ± 1.9a 58.7 ± 1.9a 53.9 ± 1.9a 16.0 ± 1.7ef 12.9 ± 1.8d 9.3 ± 1.1d17 37.8 ± 2.2c 34.0 ± 2.8c 35.1 ± 2.7c 66.0 ± 2.9a 59.8 ± 1.3a 54.9 ± 2.3a 14.4 ± 1.8g 12.1 ± 2.5d 9.1 ± 1.2d

comparisons with P-values ≥ 0.05 were considered signifi-cantly different according to Tukey test.

Results

Nitrogen metabolizing enzyme pattern during in vitroseedlingNitrate reductase (NR), Glutamate dehydrogenase(GDH) and Glutamine synthetase (GS) enzyme activi-ties were determined in leaves, stem and root parts ofin vitro-grown Argyrolobium roseum plants. Increasedpattern in the activity of NR was observed from the 1st

week until the last week of in vitro developed plants. Ac-tivity of NR raised upto 74.81% and 31.14% in in vitrogrown stem and leaves, respectively whereas, roots pre-sented 21.3% increase (Table 1). GDH showed decreasepattern during in vitro development of plants. In vitrogrown leaves showed 30.21% decrease in enzyme activ-ity, while stem and roots showed 36.56% and 37.7%decrease in pattern, respectively from the 1st week un-til 8th week. GS demonstrated an increasing pattern of

enzyme activity in in vitro plant developed on plain MSmedium. In vitro grown leaves exhibited 71.7% increasein GS activity while stem and roots resulted 238% and157% increase, respectively (Table 1).

Nitrogen assimilating enzymes pattern during callusformation and proliferationExplants of eight weeks old in vitro seedlings were cutinto small pieces and transferred to callus inductionmedium. Enzyme activities of NR, GDH and GS werestudied from 2nd week of callus formation to the 18th

week. NR presented decrease in pattern as calli devel-oped from root, stem and leaf explants. Leaf, stem androot originated calli showed 31, 21 and 28% decreasein NR activity, respectively observed from the 2nd tothe 18th week of callus formation and proliferation (Ta-ble 2). Similarly, GS also showed decline pattern duringcallogenesis. Leaf originated calli reported 56% decreasein GS enzyme level as compared to stem (45%) androots (44%). GDH presented completely reverse pat-tern in enzyme activity as compared to GS and NR.

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Table 3. Nitrate reductase (NR; µmol NO2 g fresh wt−1 h−1), glutamate dehydrogenase (GDH; µmol NAD(P)H decreased mgprotein−1 min−1) and glutamine synthetase (GS; µmol L-glutamic acid monohydroxamate/ mg protein−1 min−1) enzymes activitiesduring in vitro shoot induction of A. roseum. Results are expressed as mean ± SD of three replicates. Means sharing at least one letterare not significantly different determined by Tukey test at the 0.05 level.

NR GDH GSWeek

Leaf Stem Leaf Stem Leaf Stem

1 30.0 ± 2.9b 22.7 ± 1.9d 61.2 ± 3.0a 55.3 ± 2.4a 11.1 ± 1.1c 8.8 ± 2.6bc

2 31.1 ± 1.9b 23.0 ± 2.9c 61.0 ± 1.6a 55.0 ± 2.3a 11.4 ± 2.3c 9.0 ± 1.7bc

3 32.2 ± 2.7b 23.2 ± 2.2bc 58.4 ± 2.9ab 54.2 ± 1.0ab 12.4 ± 1.8bc 9.1 ± 1.9bc

4 33.4 ± 2.0b 23.7 ± 1.6bc 57.1 ± 2.2b 53.5 ± 2.0b 13.0 ± 1.5b 9.6 ± 2.7b5 34.0 ± 2.8b 24.0 ± 3.2b 56.1 ± 1.8b 52.8 ± 2.4b 13.2 ± 1.4b 10.0 ± 1.2b6 34.6 ± 2.6ab 24.6 ± 2.1b 55.7 ± 3.0bc 52.0 ± 3.0b 14.0 ± 1.9ab 10.0 ± 2.0b7 35.0 ± 2.9ab 25.1 ± 1.9b 55.0 ± 3.1c 51.1 ± 2.4bc 14.4 ± 2.6ab 10.2 ± 1.8b8 36.1 ± 2.0ab 26.5 ± 1.7ab 54.3 ± 2.0c 49.9 ± 2.1bc 15.1 ± 1.6ab 10.9 ± 1.8b9 38.4 ± 3.1a 28.7 ± 2.4a 54.1 ± 2.7c 49.2 ± 1.4bc 16.0 ± 1.8a 11.3 ± 2.3ab

10 39.1 ± 2.1a 29.3 ± 2.0a 53.2 ± 3.0c 48.1 ± 2.4b 16.2 ± 2.3a 12.0 ± 2.3a

Table 4. Nitrate reductase (NR; µmol NO2 g fresh wt−1 h−1), glutamate dehydrogenase (GDH; µmol NAD(P)H decreased mgprotein−1 min−1) and glutamine synthetase (GS; µmol L-glutamic acid monohydroxamate mg protein−1 min−1) enzymes activitiesduring in vitro root organogenesis of A. roseum

NR GDH GSWeek


1 44.3 ± 2.9d 34.1 ± 1.9c 38.1 ± 2.7b 51.0 ± 3.0a 46.2 ± 2.1a 40.0 ± 3.0a 18.2 ± 2.6c 13.1 ± 1.6b 10.4 ± 1.9b2 46.3 ± 1.9c 35.0 ± 2.8bc 38.3 ± 1.8b 50.7 ± 2.6a 46.0 ± 2.0a 39.5 ± 2.1a 19.1 ± 2.2c 14.0 ± 2.7b 11.9 ± 2.6b3 47.5 ± 2.7c 35.0 ± 2.0bc 39.4 ± 2.5b 50.0 ± 1.8a 45.2 ± 2.9a 38.1 ± 3.0a 20.0 ± 1.0c 14.0 ± 1.5b 12.2 ± 1.5b4 49.0 ± 2.3bc 37.0 ± 1.5b 40.0 ± 2.0ab 48.1 ± 3.0a 44.1 ± 1.8ab 37.7 ± 2.6ab 23.1 ± 1.1bc 14.0 ± 1.7b 13.0 ± 2.8b5 49.5 ± 3.0bc 38.0 ± 2.2b 41.3 ± 2.6ab 47.8 ± 2.8a 44.0 ± 2.5ab 36.6 ± 3.1ab 25.0 ± 2.1b 15.0 ± 1.1ab 14.0 ± 1.7ab

6 50.8 ± 3.2bc 39.0 ± 2.1ab 42.0 ± 2.7a 47.1 ± 1.9ab 42.0 ± 2.3b 34.2 ± 2.3b 25.1 ± 1.7b 15.6 ± 2.0ab 14.0 ± 2.3ab

7 52.9 ± 2.1b 39.1 ± 2.0ab 42.0 ± 3.0a 46.2 ± 2.6ab 41.2 ± 2.7b 33.1 ± 2.4b 26.7 ± 1.2ab 16.0 ± 2.7a 14.9 ± 1.9ab

8 53.6 ± 1.9b 39.9 ± 3.1ab 42.3 ± 2.1a 45.1 ± 2.9b 40.0 ± 3.1b 33.0 ± 2.0b 27.4 ± 2.4ab 16.1 ± 1.8a 15.3 ± 1.4ab

9 55.9 ± 1.8a 40.1 ± 2.1ab 42.9 ± 2.8a 44.9 ± 2.0b 38.8 ± 2.0b 32.9 ± 2.4b 28.7 ± 2.0a 16.4 ± 1.5a 15.9 ± 1.4ab

10 56.0 ± 2.8a 41.1 ± 2.0a 43.6 ± 1.9a 44.2 ± 1.0b 38.3 ± 2.9b 32.0 ± 2.4b 30.2 ± 1.5a 16.9 ± 1.3a 16.7 ± 2.0a

Results are expressed as mean±SD of three replicates. Means sharing at least one letter are not significantly different determined byTukey test at the 0.05 level.

Increase in pattern was noticed in calli derived fromleaf, stem and root explants. Leaf derived calli showed66% increase in enzyme activity, whereas stem and rootderived calli exhibited 100% and 292%, respectively, in-crease during callus formation and proliferation (Ta-ble 2).

Nitrogen metabolizing enzyme activities in regenerateleaves and stemShoot originated on MS medium were subjected for theanalysis of nitrogen metabolizing enzymes from the firstweek to the 10th week. NR increased almost equallyboth in stem and leaf i.e. 30% and little bit more in-crease was observed in GS; 46% in leaves and 36% instem (Table 3). Decrease (13%) in GDH was observedin both leaf and stem parts during shoot organogenesis.

Activities of nitrogen assimilating enzymes during rootdevelopment and acclimatization:During root induction and acclimatization, NR and GSincreased while GDH showed decrease in concentration.During root initiation, more increase in NR (26%) wasobserved in leaves (Table 4) as compared with root andstem however, increase in NR activity pattern changed

during acclimatization and high increase in root wasobserved (Table 5). Increase in GS was also observed inall three parts of A. roseum during root induction andacclimatization. During root induction GS activity wasobserved higher in leaves (66%) following root (59%)and stem (29%) (Table 4). However, high activity wasobserved stem part following leaf and root, respectively.Roots showed 20% decrease in GDH enzyme level dur-ing root induction and this decrease was also observedin leaf and stem parts (Table 4). The same pattern ofdecrease in GDH was noticed during acclimatizationprocess (Table 5).

Discussion

Pattern of nitrate reductase (NR) during A. roseum lifecycleNR is a light inducible enzyme and its activity re-lates with both photosynthesis and morphogenesis. NRshowed increase in activity as leaf > stem > root partsof in vitro grown A. roseum from seeds. The increasewas pronounced in photosynthetic parts as comparedwith non-photosynthetic (Fig. 1). It has been postu-lated that light acting through photosynthesis promotes

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Table 5. Nitrate reductase (NR; µmol NO2 g fresh wt−1 h−1), glutamate dehydrogenase (GDH; µmol NAD(P)H decreased mgprotein−1 min−1) and glutamine synthetase (GS; µmol L-glutamic acid monohydroxamate mg protein−1 min−1) enzymes activitiesduring acclimatization of in vitro developed plants of A. roseum. Results are expressed as mean ± SD of three replicates. Meanssharing at least one letter are not significantly different determined by Tukey test at the 0.05 level.

NR GDH GSWeek


1 57.1 ± 2.5c 42.1 ± 2.1c 44.1 ± 2.5e 43.2 ± 2.1a 36.6 ± 1.3a 30.0 ± 3.0a 31.6 ± 2.4d 16.1 ± 2.8c 16.0 ± 2.7b2 58.0 ± 2.1c 43.0 ± 1.7b 44.5 ± 3.2e 42.0 ± 1.7ab 36.0 ± 3.0a 27.9 ± 1.0b 32.0 ± 1.7cd 17.4 ± 1.9c 16.9 ± 2.0ab

3 61.8 ± 1.4bc 43.4 ± 3.0b 45.0 ± 2.2e 41.2 ± 3.0b 35.1 ± 2.1ab 27.0 ± 2.8b 32.0 ± 2.0cd 19.0 ± 2.8bc 17.1 ± 2.8ab

4 62.9 ± 3.0bc 44.0 ± 1.4b 45.1 ± 3.0e 40.9 ± 1.4b 34.2 ± 1.0b 26.3 ± 1.9bc 33.0 ± 2.8c 20.2 ± 1.5b 17.9 ± 1.4ab

5 63.4 ± 2.3b 46.9 ± 2.9ab 46.9 ± 1.9d 40.2 ± 2.9b 34.0 ± 1.4b 25.3 ± 2.8bc 35.0 ± 2.1b 20.9 ± 1.9b 18.0 ± 1.9ab

6 64.1 ± 1.9b 47.1 ± 1.1ab 48.9 ± 2.3c 40.0 ± 1.1b 32.0 ± 3.0c 24.1 ± 3.1bc 36.0 ± 1.5b 22.0 ± 1.7ab 18.9 ± 3.0a7 66.4 ± 2.7a 48.0 ± 1.9a 52.3 ± 2.8b 39.2 ± 1.9b 31.0 ± 1.5cd 22.1 ± 1.9c 37.1 ± 2.9ab 22.9 ± 2.7ab 19.1 ± 2.0a8 67.9 ± 1.4a 48.8 ± 3.0a 55.0 ± 2.0a 39.0 ± 3.0b 30.0 ± 1.6d 20.1 ± 1.3c 38.0 ± 1.6a 24.0 ± 1.9a 19.1 ± 1.7a

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

synthesis of carbohydrates; producing NADH necessaryfor NR activity (Li & Oaks 1993; Arslan & Guleryuz2005). Present study provides the evidence that NR ac-tivity amplifies while plant start photosynthesis and in-crease in biomass too.When leaf, stem and root explants inoculated on

callus initiation medium, decline in NR activity wasobserved. However, leaf explants derived calli showedzigzag pattern as compared to others (Fig. 1) mightbe due to explants; origin of calli source, the greenerand photosynthetic in nature. Leaf is the main sitefor nitrate reduction and such pattern was also no-ticed in corn leaf derived calli from 1st week to 10th

week (Remmler & Campbell 1986). The NR activitydecreased at higher rate at the end of calli prolifera-tion stage might be due to depletion of nitrate from theculturing medium and also decrease in photosyntheticactivity, the calli cells started to die.High NR activity was observed in leaves as com-

pared to stem during the regeneration phase (Fig. 1).It has been reported that photosynthetic electron flow

from PSI (photo system) and PSII are significantly im-portant in controlling the expression of NR enzyme andthere is a positive correlation between growth, proteincontent and NR activity (Dwivedi et al. 1984). Con-tinuous increase in NR pattern has also been reportedduring regeneration period of tobacco from calli (Hardy& Thorpe 1990). The regeneration phase requires moreenergy for cellular functioning, increase in biomass andalso switching towards development demands some ex-tra liabilities. This situation continued during root de-velopment, however, increase was more pronounced ascompared with shoot development. The boost might bedue to two possibilities; one refreshing the culture me-dia and second emergence of root responsible for up-take of nitrate and other nutrients. Lillo & Appenroth(2001) have reported that growth phenomenon increasethe level of nitrogen assimilating enzymes in plants.At the acclimatization stage; NR activity was max-

imum in leaf, stem and roots as compared with otherstages. This might be due to that, plants are fully devel-oped; photosynthetic activity has been started while no

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exogenous carbohydrate is available and nitrate is avail-able in the soil. Beside this, the plant has concluded thedevelopmental process and during growth period car-bon and nitrogen skeleton are required at higher level.A steady boost has been reported in tea plants duringacclimatization stage (Hajiboland et al. 2011). Lea etal. (1990) accounted same observation while correlatingplant growth and nitrogen assimilation.

Pattern of glutamate dehydrogenase (GDH) in A. ro-seumDuring the growth process from seedling to plant; de-crease in GDH activity was observed in all plant parts.Normally, catabolic enzymes concentrations remain lowduring growth and developmental processes. Decline inammonia and sucrose concentration with time in theculture medium might also be responsible for low GDHlevel in A. roseum. NAD(P)H GDH enzyme is moreresponsive to stress conditions as compared to NADHGDH and is localized in chloroplast of photosynthesiz-ing tissues (Lam et al. 1996). Miyashita & Good (2008)also suggested that under carbon deficiency, NAD(H)-GDH involves in the breakdown of amino acids andthe supply of respiratory substrate to the tricarboxylicacid cycle because GDH do not require ATP for itsfunctioning while parallel enzyme, GS-GOGAT is ATP-dependent (Fischer & Klein 1988).GDH started to increase as calli started to develop

from explants and the raise continued during prolifera-tion stage. At the end of proliferation phase, leaf initi-ated calli showed only 66% increase from starting stageas compared to stem (100%) and root (292%). Manyfold increase in GDH activity in root explants derivedcalli might be due to source and biochemical charac-teristics of explants. Excess of ammonia (20.1 mM) inthe medium also enhances GDH activity used to in-corporate ammonium into amino acids (Kormutak &Vookova 1997). Excess ammonia triggers the α-subunitof GDH, which involves in the synthesis of proteins andincreased activity of GDH may also be due to de novoprotein synthesis (Loulakakis & Roubelakis-Angelakis1991). GDH also plays an important role in NH4 as-similation and alleviating the toxicity of excessive NH4from tissue (Afzal et al. 2006). Callus culture containedalmost the double of GDH enzyme activity as comparedto explants initiated for calli origination. This deter-mines a radical change in overall metabolic behavior ofcallus. GDH enzyme is mainly localized in the phloemcells (Dubois et al. 2003; Tercé-Laforgue et al. 2004;Fontaine et al. 2006; Fontaine et al. 2012), but its syn-thesis enhance in the mitochondria, when the ammo-nium concentration increase above a certain threshold(Tercé-Laforgue et al. 2004).During shoot regeneration from calli, GDH activity

continued to decrease and this reduction preceded evenduring root development and acclimatization stages.The catabolic enzymes normally tend to decrease dur-ing the developmental process. Regeneration of shootsand roots are organ development and requires aminoacids 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 acidmetabolism and in particular metabolism derived fromglutamate and glutamine, which could greatly influ-ence the translocation and use of amino acids thatcan act as nitrogen transport molecules during plantgrowth and development (Lea & Azevedo 2007; Lea etal. 2007). This nutritional metabolic shift up duringcallogenesis and shift down for the period of organo-genesis influenced by the nutrients and phytohormonesin the medium might also be responsible for decrease inGDH (Soulen & Olsan 1969). According to Loulakakis& Roubelakis-Angelakis (1991) excess ammonia is alsoresponsible for the increased activity of NADH-GDH inleaves, shoots and roots. Glutamate is a primary prod-uct of ammonia assimilation in photosynthetic tissues,although other amino acids such as alanine and asparticacid have been demonstrated to be important productswhen nitrate is utilized as nitrogen source.

Pattern of glutamine synthetase (GS) in A. roseumOne of the most important enzymes in nitrogen fix-ation and ammonia incorporation into protein is GS(Magalhaes & Huber 1991). Leaf showed high level ofGS activity as compared to stem and roots during invitro germination of A. roseum. Such increase in ac-tivity has also been reported in other plants (Mack &Schjoerring 2002). This might be due to that plants as-similate most of their nitrogen in the leaves and duringthe growth period and for normal growth, activities ofbiosynthetic 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 ofGS in cells grown on NH4 containing medium has beenreported by several authors (Robinson et al. 1992; Flo-rencio & Ramos 1985). Ammonium and nitrate uptakesand their metabolism in cells significantly determine thebiochemical characteristics of the callus. Leaf derivedcalli reported fast decline as compared to the stem androot. This might be due to the fact that source of calliformation plays an important role in the biochemicaland physiological characteristics of calli. At the end ofproliferation phase, no drastic difference in the activ-ity of GS was observed in leaf, stem and root derivedcalli. Calli absorb nitrate from the medium which re-sult excess of ammonia at the lateral stage. It has beenreported that excess of ammonia inhibits GS activity(Kormutak & Vookova 1997). Furthermore, decreasedpattern of GS might also be due to the medium whichis exhausted by various nutrients especially sucrose andpresence of phytohormones play synergistic role control-ling the enzymatic activities.Reverse pattern was observed by GS enzyme dur-

ing regeneration phase as compared to callogenesis.This increase in pattern continued even during rootdevelopment (Fig. 1). The activity of biosynthetic en-zymes increases during the regeneration phase andplant growth regulators enforce the callus to regener-ate shoot; might be involved in regulation of certain

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484 D. Habib et al.

enzymes (Sood et al. 1996). Chanda et al. (1998) hassuggested that that kinetin treatment initially showeda slight promotion in cytosolic GS of mustard seedlingshypocotyls. During organogenesis, GS plays an impor-tant role in nitrogen metabolism, by enhancing theamides and amino acids synthesis required for proteinsynthesis resulting high protein content in regeneratedtissues as compared to callus (Miflin & Habash 2002).Based on our experiments we can conclude that

an xtensive reverse pattern was shown by nitrogen as-similating enzymes during regeneration as comparedto callogenesis process. Activity of GDH started de-creasing as regeneration process initiated, while GS andNR showed ascending pattern as developmental pro-ceeded. Catabolic enzymes show descending patternduring organogenesis process. Initiation of organizeddevelopment involves a shift in metabolism, which pre-cedes and is coincident with the shoot development pro-cess (Thorpe 1983). This pattern continues in acclima-tization stage shown by GDH, GS and NR. GS and NRare known for their assimilatory role in plant develop-ment.

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Received November 21, 2014Accepted February 12, 2015

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36 Response of nitrogen assimilating enzymes during in vitro culture of Argyrolobium roseum