Journal of Plant Physiology & Pathology ISSN: 2329-955X

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Research Article, J Plant Physiol Pathol Vol: 8 Issue: 3

Regulation of Glutamine Synthetase (GS) Activity and Root Morphological Plasticity in the Nitrogen Fixing Azolla- Anabaena Symbiont under Nitrogen Limiting Conditions

Yusuf A1*, Aparna MB1 and Heimer YM2

1Interuniversity Centre for Plant Biotechnology, Department of Botany, University of Calicut, Thenhipalam 673635, Kerala, India

2Albert Katz Centre for Desert Agrobiologies, Ben Gurion University of the Negev, sede Boquer Campus, Israel

*Corresponding Author : Yusuf A
Interuniversity Centre for Plant Biotechnology, Department of Botany, University of Calicut, Thenhipalam 673635, Kerala, India
E-mail: [email protected]

Received: September 28, 2018 Accepted: October 20, 2018 Published: October 29, 2018

Citation: Yusuf A, Aparna MB, Heimer YM (2018) Regulation of Glutamine Synthetase (GS) Activity and Root Morphological Plasticity in the Nitrogen Fixing Azolla-Anabaena Symbiont under Nitrogen Limiting Conditions. J Plant Physiol Pathol 6:5. doi: 10.4172/2329-955X.1000191


The present study was conducted to identify the role of the endosymbiont, Anabaena azollae in fixing atmospheric N2 in the Azolla-Anabaena symbiosis. The Azolla-Anabaena azollae (association) grown in nitrogen-free modified Hoagland’s medium supplemented with 10% (v/v) N2 using an artificial gas mix, resulted in the increase in root number and root length. The GS specific activity of the association was 75 ± 6 nmole γ-glutamate mg protein-1 min-1 and the total N content was lesser in cultures grown in 10% (v/v) N2. Similarly, the artificially produced Anabaena free Azolla (endophyte-free), developed increased root length, root number and reduced GS specific activity (30 ± 10 nmole γ-glutamate mg protein-1 min-1), which on supplementation with 2.5 mM NH4NO3 did not show any change in the root length, root number and GS activity. However, the association cultured in 10% (v/v) N2+2.5 mM NH4NO3, reversed the effect of 10% (v/v) N2 treatment exhibited a GS specific activity of 295 ± 120 nmole γ-glutamate mg protein-1 min-1 and the morphological features were similar to the control plants. The western blot analysis of the GS protein from the association showed two isoforms of GS (GS1 and GS2) of equal abundance under all growth conditions, whereas, one diffused band of intermediate size between GS1 and GS2 was observed in the endophyte-free fern.

Keywords: Azolla anabaena azollae; Symbiotic N2 fixation; Endophyte-free fern; Glutamine synthetase activity


Azolla-Anabaena azollae association is an obligatory symbiotic system between the water-floating fern Azolla and the N2 fixing cyanobacterium Anabaena azollae. Azolla-A. azollae symbiosis is the only known mutual symbiosis between a pteridophyte and a diazotrophic prokaryote. The symbiosis of Azolla-Anabaena represents one finest example of associations which can fix atmospheric nitrogen (N2) [1]. In the association, the cyanobacterial symbiont provides the Azolla with nitrogen nutrition enabling the fern to grow in a nitrogen-free nutrient medium [2]. Anabaena provides ammonia to the fern, and the fern, in turn, supplies the cyanobacterium with photosynthetic assimilates [3] thus helps to maintain the C-N balance in the association. Anabaena converts fixed N2 to ammonia assisted by nitrogenase enzyme and approximately one-half of the nitrogen fixed by the algae is liberated as ammonium ion. As the nitrogen-fixing activity of the association is performed entirely by the symbiont, the cyanobiont-free fronds are incapable of nitrogen fixation, resulting in their failure to survive in nitrogen-free medium.

In the association, the fixed N2 is converted to ammonium ions and are assimilated by the enzyme glutamine synthetase and converted to amino [4]. Glutamine synthetase (GS; EC is the key enzyme involved in the assimilation of inorganic nitrogen into organic forms [5], catalyzes the incorporation of ammonium into glutamate and generates glutamine in an adenosine triphosphate (ATP)-dependent reaction [6], which provides nitrogen groups, either directly or via glutamate for the biosynthesis of all nitrogenous compounds to the plant [7]. GS exists in two isoforms, GS1 and GS2, having different functions and organellar localizations. GS1 is located in the cytosol and nonphotosynthetic portion of the cells whereas GS2 is seen in the chloroplasts [8].

Anabaena free Azolla (Endophyte-free) is non-existing in nature, but can be produced in the laboratory by curing the symbiont from the fern. Endophyte-free fern has been artificially produced by chemical or antibiotic treatments, or by exposure to adverse environmental conditions [9]. Endophyte-free fern requires combined nitrogen supplemented growth medium, however, its growth is very slow. The endophyte-free plants grow well and sporulate on soil-water medium when supplemented with small quantities of nitrogenous substances [10].

Nitrogen is one of the major factors for plant growth and its deficiency causes changes in growth and development. To overcome N deficiency, plants induce signals in the leaves and transfer it to the roots for effective absorption of soil N to balance the C-N content. Plants have the ability to acclimatize themselves to reduced nitrogen by increasing the nutrient acquisition owing to altered morphology, root/shoot ratio and metabolism [11]. In this study, the response of the Azolla-Anabaena association to N limiting conditions was investigated by growing Azolla-Anabaena association in an artificial gas mixture with low nitrogen and also by using artificially created endophyte-free fern. Growth parameters were analyzed with respect to morphological changes and the growth characteristics were correlated with the presence/absence of endophyte in Azolla-Anabaena association under nitrogen limiting conditions.

Materials and Methods

Plant material

A. pinnata fronds were collected from the Botanical garden, University of Calicut. Adhered green algae and other microorganisms were removed by washing the fronds thoroughly with water for several times, followed by surface sterilization with 0.12% (v/v) sodium hypochlorite solution containing few drops of Tween 20 for 10 mins. The fronds were then washed with sterile distilled water six times, blotted dry and transferred to Hoagland’s nutrient medium [12].

Endophyte-free Azolla

Endophyte-free A. pinnata cultures were produced by treating the frond shoot apex with 0.5% streptomycin sulphate for 5 min and transferred to modified Hoaglands medium with 2.5 mM NH4NO3. The cultures were crushed after 21 days of growth and observed un

Culture medium and establishment of N limiting growth conditions

Modified Hoagland’s medium with or without combined nitrogen was used as the culture medium for growing A. pinnata fronds. The cultures were kept in 16-hr light, with a light intensity of 1500 μEm-2s-1 and a temperature of 25 ± 2°C. Illumination was provided by cool white fluorescent lights. 10% (v/v) N2 was supplied to the cultures using an artificial gas mixture containing 10% (v/v) N2, 20% (v/v) O2, 0.035% (v/v) CO2 and 69.965% (v/v) Argon. A pinnata fronds were cultured in Hoagland’s medium with the following growth conditions 1) Air, 2) Air+2.5 mM NH4NO3 3) 10% (v/v) N2 gas mixture 4) 10% (v/v) N2 gas mixture+2.5 mM NH4NO3 and the growth parameters were compared.

Doubling time of A. pinnata

The growth rate of Azolla cultures was measured by calculating the doubling time using the formula derived by Aziz and Watanabe [4]. The cultures survived in the medium till the 24th day of culture and started shedding their roots from the 17th day onwards.

Doubling time=t/r

Where, t=duration of growth

r=[log (Wt/Wo)]/0.301

Wt=Weight of Azolla after‘t’ days

W0=Weight of initial inoculum

Root number and length

The root number and root length were measured from each experiment by counting individual roots of randomly selected fronds. The root length was measured by placing the representative fronds on a graph paper, and an average of at least 50 roots was measured.

GS extraction and transferase assay

GS transferase assay was carried out according to the protocol explained by Shapiro and Stadtman [13] 100 mg of A. pinnata fronds from each experiment were powdered separately in liquid nitrogen, emulsified in an extraction buffer containing 100 mM Tris HCl (pH 7.2), 3 mg DTT, 0.2 g PVP, and 0.2 ml PMSF using a mortar and pestle kept on ice. The homogenate was then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected for GS transferase assay and protein quantification. The reaction mixture contained 0.25 ml reaction buffer (made of 40 μmol Tris-HCl pH 7.5; 30 μmol glutamine (pH 7.0); 3 μmol MnCl2; 0.4 μmol ADP; 20 μmol K-arsenate and 60 μmol hydroxylamine which was neutralised with 60 μmol NaOH before addition to the reaction mixture), 0.15 ml double distilled water and 0.1 ml supernatant. The reaction mixture was incubated at 30°C for 20 min in a shaking water bath. The reaction was stopped by adding 1 ml stop solution containing 10% (w/v) FeCl3 in 0.2 N HCl, 24% (v/v) TCA and 54% (v/v) HCl. The optical density was measured at 540 nm against blank to which the stop solution was added prior to addition of enzyme extract and incubated at 30°C for 15 min.

Total protein was quantified according to Lowry et al. [14] using bovine serum albumin (BSA,1 mg/ml) as standard. The concentration of γ-glutamyl hydroxamate and protein was calculated from their corresponding standard graphs and the specific activity of GS was calculated.

Total Kjeldahl Nitrogen

Total nitrogen content from A. pinnata grown under different experimental conditions was measured using Kjeldahl method [15]. Powdered plant tissue (0.5 gm) was taken in a Kjeldahl flask and 5 g K2SO4, 0.5 g CuSO4, and 20 ml H2SO4 was added. The mixture was slowly shaken, heated at 60°C and boiled for 1½ hrs. After cooling, 150 ml 30% NaOH solution was added and distilled for 20 min. The vapours were collected in 20 ml 4% (w/v) boric acid and titrated using methyl red as an indicator. The percentage of nitrogen was calculated by the following equation.

% Nitrogen=(ml standard acid-ml blank) x N of acid x 1.4007/ Weight of sample in grams

Total carbon

Total carbon of A. pinnata grown under different experimental conditions was estimated using CHNS analyser (Model: Elementar Vario EL III). Dried samples (1.5-2.0 g) were ground to make a fine powder using mortar and pestle and subsequently measured at 980°C using sulfanilic acid as standard.

Protein precipitation, PAGE and Western blot analysis

Total protein was concentrated by treating the supernatant with 0.1 ml of 0.15% (w/v) Deoxycholate, mixed and kept on ice for 10 min. 0.1 ml of 72% (v/v) TCA was added, mixed and centrifuged at 10000 rpm for 15 min. The pellet was washed with 25% (v/v) acetone and dissolved in sample buffer. SDS-PAGE was conducted using a mini vertical slab gel (8 × 7 cm). 50 μg of the purified protein from different N treatments and endophyte free were mixed with sample buffer in a 1:1 ratio and heated in a boiling water bath for 3 min. The samples were loaded to the gel and electrophoresed for 1½ hrs. After electrophoresis, the proteins bands were blotted to nitrocellulose membrane using a protein blotting unit. The blotted membrane was first blocked with skimmed milk and then treated with anti-GS monoclonal antibody solution in Tris Buffer Saline (TBS) for 1 h then washed thrice with TBST. After incubation with secondary antibody (Alkaline phosphatase) in TBS, the membrane was again washed thrice with TBST and finally with TBS for 5 min. Color was developed by the color development solution containing the developer and 200 μl Nitro Blue Tetrazolium and 100 μl BCIP (5-bromo-4-chloro-3-indolyl phosphate) for 45 min and finally washed with stop solution (saturated BCIP solution) to develop color. The bands developed were observed.

Statistical analysis

Results were analysed using one-way analysis of variance (ANOVA). ANOVA was performed by considering each experiment as an independent unit using SPSS software version16.0. As significant variations were observed in ANOVA the values were compared using Duncan’s multiple range test at 5% probability level.


Effects of N deprivation on the morphological parameters

Doubling time: The A. pinnata cultures showed variability in doubling time with respect to the N content in the growth medium. The A. pinnata association grown in ambient air showed a doubling time of 4.26 days, upon the addition of 2.5 mM NH4NO3 the doubling was reduced to 4.04 days. Association grown in 10% (v/v) N2 required a higher doubling time of 7.34 days which was reduced to 5.03 days when supplied with 2.5 mM NH4NO3, however, the endophyte-free showed a doubling time of 8.95 days (Figure 1).

Figure 1: Doubling time of A. pinnata association grown in different N conditions and endophyte free A. pinnata grown in 2.5 mM NH4NO3. The values are mean ± SE, N=3). The values are mean ± SE and N=3. Post hoc test using Duncan’s multiple range test at p ≤ 0.05 indicated significant differences among the treatment as shown by different letters.

Morphology of roots under different N2 concentrations

The fronds of the association showed compactly arranged leaves and small sized lesser roots, with few lateral roots however, upon treatment with 2.5 mM NH4NO3 the root growth was reduced and few small roots were produced. The association grown in medium without added N, under ambient condition has much longer roots than cultures grown in 2.5 mM NH4NO3. The endophyte free A. pinnata grown in N free medium turned yellow after 5 to 6 days and failed to survive. Root length, root number and root hair development varied with the N concentrations. The number of roots, lateral roots and root length increased, when the association was grown in 10% N2, however when supplemented with 2.5 mM NH4NO3 the root morphological changes were reversed and the root length reduced with much less lateral roots and the fronds started to turn dark green.

Plants grown in 10% N2 showed a mean root length of 1882 ± 10 mm. The root length of plants grown in different conditions such as control,+2.5 mM NH4NO3, and 10% (v/v) N2+2.5 mM NH4NO3 were 461 ± 30, 557 ± 18, and 533 ± 15 mm respectively (Figure 2 and Figure 3). The result suggests that the plants subjected to nitrogen limitation developed a large number of roots with increased length and provision of combined nitrogen reversed these effects. The endophyte free A. pinnata became pale green in colour, developed a massive root system with lots of lateral roots even in presence of supplied N, suggesting their inability to utilize N when compared to the association (Figure 4).

Figure 2: Root morphology variations in the association grown in Hoagland’s medium containing 2.5 mM NH4NO3 and in reduced atmospheric N2.

Figure 3: The differences in root number in A. pinnata cultures treated with different N sources. The values are mean ± SE, N=3. The values are mean ± SE and N=3. Post hoc test using Duncan’s multiple range test at p ≤ 0.05 indicated significant differences among the treatment as shown by different letters.

Figure 4: Root morphology variations in endophyte free Azolla (a) and the association (b) grown in Hoagland’s medium containing 2.5 mM NH4NO3.

GS Specific activity

In 10% N2 containing medium, the GS specific activity of the association was 75 ± 6 nmole γ-glutamate mg protein-1 min-1, however, the addition of 2.5 mM NH4NO3 increased the GS specific activity to 295 ± 120 nmole γ-glutamate mg protein-1 min-1. Cultures grown in ambient condition, supplemented with 2.5 mM NH4NO3 showed a higher GS specific activity of 400 ± 90 and 510 ± 55 nmole γ-glutamate mg protein-1 min-1 respectively. The endophyte free cultures showed low GS specific activity of 30 ± 10 nmole mg protein-1 min-1, a result similar to the nitrogen limiting condition even provided with combined nitrogen in the form of NH4NO3 (Figure 5).

Figure 5: GS specific activity (nmole γ-glutamate mg protein-1 min-1) in different culture conditions in the association and in endophyte free. The values are mean ± SE, N=3. Post hoc test using Duncan’s multiple range test at p ≤ 0.05 indicated significant differences among the treatment as shown by different letters.

Total carbon and nitrogen

Total carbon and nitrogen analysis of A. pinnata under all the experimental conditions showed that the total N% of endophyte free Azolla was the lowest and even the association treated with 10% N2 showed an N% of 3.22. Association under ambient conditions had 5.48% N and a C/N ratio of 7.92. The endophyte-free cultures as well as cultures kept in 10% N2 had higher C:N content (Figure 6).

Figure 6: Total carbon, nitrogen and C-N ratio of Azolla-anabaena association and endophyte free Azolla under different experimental conditions. The values are mean ± SE, N=3. Post hoc test using Duncan’s multiple range test at p ≤ 0.05 indicated significant differences among the treatment as shown by different letters.

The western blot analysis of GS protein using anti GS monoclonal antibody showed two specific bands of GS1 and GS2 in A. pinnata association in all experimental conditions like ambient air, air+2.5 mM NH4NO3, 10% N2 and 10% N2+2.5 mM NH4NO3. Whereas, in endophyte free only one diffused band of intermediate size between the two GS isoforms was observed (Figure 7), suggesting that even though the GS protein is present its isoenzymes are not functional in endophyte free fern.

Figure 7: Western blot assay of GS isoenzymes from Azolla-Anabaena association and endophyte free Azolla under different experimental conditions.


Azolla-Anabaena azollae, a symbiotic N2 fixing association under N stress developed a massive root system. The root length of the association under N limitation increased three fold compared to ambient N2. The root length statistics showed that plants exposed to 10% (V/V) N2 showed an increase in biomass and a large number of roots with a lot of lateral roots and combined N supplementation reversed the effect. Plants show developmental plasticity, thus has the ability to modify their root architecture, stem leaf modifications in order to absorb the available N [16]. Various adaptive mechanisms are developed in plants in order to assimilate N from the field. Plants adapt themselves to acquire soil nitrogen by changing the root morphology such as root radius, root length, and root surface area and also by increasing the quantity of biomass allocated to roots [17], the assimilation of soil N by plants is more dependent upon root length or root surface area than total root biomass [18]. Bonifas and Lindquist [19], reported various adaptive mechanisms in velvet leaf to increase N uptake under limiting conditions. Nutritional deficiency induced modifications included, increasing the quantity of biomass allocation to roots, altering root morphological characteristics, increasing the N uptake efficiency of roots, or their combination, thus advocating the effects obtained in the Azolla-Anabaena symbiosis for want of N supply to the plants, thereby increasing the biomass.

Antibiotic treatment eliminated the algal colonies from Azolla, thus providing the Anabaena free culture. The removal of the algal colony from the association increased the doubling time [20]. Azolla treated with erythromycin did not respond after 8-10 days and showed senescence symptoms due to its toxic effect on the cyanobacterium. The plants initiated growth only after 15 days when transferred to a nitrogen-containing medium and the leaf cavities were devoid of cyanobacterium after antibiotic treatment. The cyanobacteria were eliminated from the leaf cavities when treated with Novobiocin to obtain Anabaena free Azolla [20]. The cyanobacterium free cultures of Azolla, are devoid of N2 source and behave identically with the artificially N limitation condition.

GS plays a key role in the assimilation of ammonia to glutamine, which is the main form of organic nitrogen for transport through vascular tissues [21]. Azolla assimilates fixed nitrogen mainly via the glutamine synthetase cycle with little or no contribution of glutamine dehydrogenase [22]. The GOGAT enzyme is second in the metabolic sequence of ammonia assimilation since GS provides GOGAT with its substrate, glutamine. Thus the GS enzyme is the one that responds rapidly to changes in nitrogen nutrition levels. Although GOGAT provides the glutamate required by GS, other enzymes including GDH, transaminase and glutaminase are able to catalyze reactions that synthesize glutamate, while GS is the primary enzyme catalyzing glutamine synthesis [23]. Glutamine synthetase (GS; EC assimilates ammonium into glutamine, utilised for the biosynthesis of all essential nitrogenous compounds [24]. GS activity is upregulated during the supply of N compounds to the root system and is affected by the external NH4+ concentration. The association treated with 10% N2 and Anabaena free Azolla, deprived of N showed a lesser GS specific activity when compared to those plants kept under ambient atmospheric conditions and with combined nitrogen. The dependence of GS activity on the nitrogen source for the growth and the nutritional status of the cells are reported in Agmenellum quadruplicatum strain PR-6 [25]. In the association the absorbed nitrogen is converted into ammonium in the heterocyst of Anabaena accounts for the increased GS activity compared to the Anabaena free Azolla. The absence of Anabaena affected the ammonium availability and therefore showed a lesser GS activity in endophyte free Azolla, even though the GS protein is present in the endophytefree, probably the post-translational modifications of GS protein is hampered in endophyte-free, accounting for the defused bands seen in the Western blot assay.

Total nitrogen in the plants, whether derived from nitrate, ammonium, through N2 fixation, or catabolism of proteins, is channeled through reactions catalyzed by GS. Accordingly, GS plays a vital role in nitrogen metabolism of vascular plants and is a major checkpoint controlling plant growth and productivity [26]. GS presents two isoforms in higher plants which are located in the cytosol (GS1) and in the plastids (GS2) and are encoded by a small multigene family. The subunits of cytosolic and plastid located GS differ in molecular mass and can be readily separated by SDS-PAGE [27]. The western blot assay with anti-GS monoclonal antibody provided two specific GS bands in the association whereas the endophyte free produced only a diffused band. The diffused GS band in endophytefree Azolla indicates that the expression of GS in Azolla-Anabaena association is influenced by the endophyte. The proper expression and functioning of GS require the presence of the endophyte, assisting the post-translational modification of GS. These results are in accordance with the studies that the hair cells of cyanobiont containing plants had higher levels of GS1 and GS2 than cyanobiont free plants [28]. The GS isozymes have different metabolic roles, and their activities vary with plant development in different organs and cell types [29]. GS2 is the predominant isozyme in leaf mesophyll cells, where it assimilates ammonia originating from nitrate reduction and photorespiration [30,6]. GS1 has multiple metabolic functions, such as primary ammonium assimilation in the roots, and catabolised ammonia reassimilation for transport and distribution all through the plant, and localizes to the vascular cells of different tissue of Arabidopsis [31], wheat (Triticum aestivum L.), rice (Oryza sativa), tobacco (Nicotiana tabacum) [32], and potato (Solanum tuberosum) [33]. The expression of GS isoform genes has been reported to be regulated by external factors such as light, the nitrogen source and the symbiotic association with Rhizobium [34] or by several metabolites including amino acids and soluble carbohydrates [35] in higher plants. Thus it is clear that there is a cross-talk between the endophyte and Azolla which leads to the regulation of GS expression.


The study proved that in the endophyte-free fern, even with N supply cannot utilize the N properly due to its lack of GS enzyme resulting in chronic nitrogen limitation. The effects of the association under N stress mimics morphologically and biochemically the endophyte-free proving that the endophyte is essential to establish a cross-talk between Azolla and Anabaena.


The authors are grateful to the Director, Interuniversity Centre for Plant Biotechnology, Department of Botany, and University of Calicut for providing the facilities.


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