Lipid peroxidation and antioxidant enzyme activities ...

Environmental and Experimental Botany 58 (2006) 93–99

Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots Y.H. Dewir a,b , D. Chakrabarty a,c , M.B. Ali a , E.J. Hahn a , K.Y. Paek a,∗ a

Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheong-ju 361-763, Republic of Korea b Department of Horticulture, Faculty of Agriculture, Tanta University, Kafr El-Sheikh 33516, Egypt c Floriculture Section, National Botanical Research Institute, Lucknow, India Accepted 27 June 2005

Abstract A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H2 O2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS. © 2005 Elsevier B.V. All rights reserved. Keywords: Antioxidant enzymes; Euphorbia millii; Hyperhydricity; Glutathione

1. Introduction Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Losses of up to 60% of cultured shoots or plantlets have been reported due to hyperhydricity in commercial plant micropropagation (Pâques, 1991), which reflects the importance of this problem. The phenomenon has been correlated to water availability, microelements and/or hormonal imbal∗

Corresponding author. Tel.: +82 43 261 3245; fax: +82 43 272 5369. E-mail address: [email protected] (K.Y. Paek).

0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2005.06.019

ance in the tissue culture medium (Kataeva et al., 1991). In addition to a high water content in leaf and stem tissues (Pâques and Boxus, 1987), vitrified plantlets have poor epicuticular wax production (Majada et al., 2001). Hyperhydric tissues usually show a broad array of side effects, i.e. anoxia and mineral deficiencies (Olmos and Hell´ın, 1998), alterations of peroxidase, PAL and ACC-synthase activities, and failure in lignin biosynthesis (Kevers et al., 1984; Olmos et al., 1997a, b). However, it remains difficult to determine whether hyperhydric shoots (HS) are stressed or not. Undoubtedly, a better understanding of the physiological bases of hyperhydricity in relation to its stress

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response would be of great interest in order to prevent this abnormality. High relative humidity has been considered to be one of the most important environmental factors responsible for hyperhydricity in plants cultured in vitro which is associated with reduced transpiration and excessive water uptake that could reduce the level of oxygen within tissues to nearhypoxia, which interferes with respiration at the level of electron transport. Under hypoxia conditions, some of the metabolic activities that can generate H2 O2 in plants could be disrupted, with the production of toxic levels of H2 O2 and generation of oxidative stress (Asada and Takahashi, 1987; Olmos et al., 1997a, b). In plant cells, the oxidative stress reactions are associated with toxic free radicals from the reduction of molecular oxygen to superoxide radicals, singlet oxygen, hydroxyl radicals and hydrogen peroxide. To counter the hazardous effects of ROS under stress, plants have evolved a complex antioxidative defense system composed of both antioxidant enzymes and metabolites such as APX, CAT, SOD, GR, ASA, GSH, GSSG and Vitamin E (Ahmed et al., 2002; Sairam et al., 1998, 2002). In order to precise these hypotheses and to understand more precisely stress and morphological responses in HS, the main objectives of this work were to examine whether the oxidative stress is involved in hyperhydricity. Therefore, we have assessed the level and changes in activities and isoenzyme patterns of antioxidative enzymes in hyperhydric Euphorbia plants. 2. Materials and methods 2.1. Plant material In vitro regenerated plantlets from inflorescence of Euphorbia millii, maintained onto MS basal medium (Dewir et al., 2005), were used for this experiment. Apical meristems (2.5–3.0 cm) from in vitro culture (30 explants per bioreactor) were transferred to 5 l balloon type bubble bioreactor (BTBB) with 1.5 l MS liquid medium supplemented with 3% sucrose, 1.0 mg l−1 BA + 0.3 mg l−1 IBA. The pH of the medium was adjusted to 5.8 before autoclaving (at 121 ◦ C and 1.2 kg cm−2 pressure for 30 min). The temporary immersion bioreactor system was programmed to immerse the plantlets in medium for four times per day and 30 min every time. All the bioreactors were maintained at 25 ◦ C under 50 ␮mol m−2 s−1 PPF under 16 h photoperiod per day. A large number (30%) of micropropagated E. millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. These hyperhydric leaves as well as normal in vitro leaves were selected as an experimental material. 2.2. Antioxidant enzyme assay For determination of antioxidant enzyme activities, 0.5 g of leaves was homogenized in 1.5 ml of respective extraction

buffer in a pre-chilled mortar and pestle by liquid nitrogen. The homogenate was filtered through four layers of cheesecloth and centrifuged at 22 000 × g for 20 min at 4 ◦ C. The supernatant re-centrifuged again at 22 000 × g for 20 min at 4 ◦ C for determination of antioxidant enzyme activities. The preparation was applied to a column of sephadex G-25, equilibrated with the same buffers and kept in an ice bath until the assays were completed. Protein concentration of the enzyme extract was determined according to Bradford (1976). Superoxide dismutase (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT) according to the method of Beyer and Fridovich (1987). Leaves were homogenized in 1 ml cold 100 mM K-phosphate buffer (pH 7.8) containing 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1% (w/v) polyvinyl-pyrrolidone (PVP) and 0.5% (v/v) Triton X100. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. For the determination of APX, MDHAR and DHAR activities, Leaves were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 5 mM ascorbate, 10% glycerol and 1 mM EDTA. APX (EC 1.11.1.11) activity was determined in 1 ml reaction mixture containing 50 mM K-phosphate (pH 7.0), 0.1 mM ascorbate (extinction coefficient, 2.8 mM−1 cm−1 ), 0.3 mM H2 O2 . The decrease in absorbance was recorded at 290 nm for 3 min (Chen and Asada, 1989). MDHAR (EC 1.6.5.4) activity was assayed following the decrease in absorbance at 340 nm due to NADH oxidation using an extinction coefficient of 6.22 mM−1 cm−1 (Hossain et al., 1984). The 1.0 ml reaction mixture consisted of 90 mM K-phosphate buffer (pH 7.0), 0.0125% Triton X-100, 0.2 mM NADH, 2.5 mM L-ascorbic acid, and required amount of enzyme extract. One unit of asccorbate oxidase is defined by the manufacturer (units as defined by Sigma Chem. Co.) as the amount that causes the oxidation of 1 ␮mol of ascorbate to monodehyadroascorbate per minute. DHAR (EC 1.8.5.1) activity was measured by measuring the reduction of dehydroascorbate at 265 nm for 4 min (Doulis et al., 1997). The 1.0 ml reaction mixture contained 90 mM K-phosphate buffer (pH 7.0), 1 mM EDTA, 5.0 mM glutathione (GSH), and required amount of enzyme extract. The reaction was initiated by the addition of 0.2 mM dehyadro ascorbate (DHA) (extinction coefficient, 14 mM−1 cm−1 ). For determination of CAT, POD, GR, GPX and GST, leaves were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA under liquid nitrogen. Catalase (EC 1.11.1.6) activity was determined by following the consumption of H2 O2 (extinction coefficient, 39.4 mM−1 cm−1 ) at 240 nm for 3 min (Aebi, 1974). G-POD (EC 1.11.1.7) activity was measured by following the change of absorbtion at 436 nm due to guaiacol oxidation (extinction coefficient, 6.39 mM−1 cm−1 ) following Pütter (1974). The activity was assayed for 5 min in a reaction solution composed of 50 mM K-phosphate buffer (pH 7.0), 20.1 mM guaiacol, 12.3 mM H2 O2 and required amount of enzyme extract from leaves. GR (EC 1.6.4.2) activity was assayed


by following the reduction of DTNB at 412 nm (extinction coefficient, 13.6 mM−1 cm−1 ) with some modifications as described by Smith et al. (1988). The assay mixture (1 ml) contained 100 mM K-phosphate buffer (pH 7.5), 1 mM oxidized glutathione and 0.1 mM NADPH and 100 ␮l of enzyme extract. GST (EC 2.5.1.18) activity was determined by measuring the increase in absorbance at 340 nm (extinction coefficient, 9.6 mM−1 cm−1 ), incubating reduced glutathione (GSH) and 1-chloro-2, 4-dinithrobenzene (CDNB) as substrates, according to Droter et al. (1985). The 1 ml reaction mixture contained 100 mM K-phosphate buffer, pH 6.25 and 0.8 mM 1-chloro-2,4-dinitrobenzene (CDNB). GPX activity was assayed by the oxidation of NADPH at 340 nm (extinction coefficient, 6.22 mM−1 cm−1 ) as described by Pagila and Valentine (1967). The reaction mixture constituted of 50 mM K-phosphate buffer, pH 7.0, containing 1 mM EDTA, 0.24 unit GR (EC 1.6.4.2; Sigma–Aldrich, St. Louis, USA), 10 mM GSH, 0.20 mM NADPH, and 1 mM sodium azide. After addition of enzyme, test tubes were incubated at 37 ◦ C for 10 min. The reaction was initiated by addition of 1 mM H2 O2 .

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(pH 7.8) containing 28 mM TEMED and 2.45 mM NBT for 10–20 min with gentle agitation in the presence of light. SOD activity was detected by the procedure described by Beauchamp and Fridovich (1971). The gel equilibrated with 50 mM K-phosphate buffer (pH 7.8) containing 2.8 × 10−5 M riboflavin, 0.028 M N,N,N ,N -tetramethyl ethylenediamine (TEMED) for 30 min. The gel was washed in distilled water for 1 min and submerged in a same solution (mentioned above) containing 2.45 mM NBT for 10–20 min with gentle agitation in the presence of light, the enzymes appeared as colourless bands in a purple background. CAT activity was detected following the procedure of Woodbury et al. (1971). The gel was incubated in 0.01% H2 O2 for 10–15 min and washed with distilled water twice and incubated for 15–20 min in 1% FeCl3 and 1% K3 [Fe(CN6 )]. After staining gel were washed carefully with tap water. For POD, gel was incubated in 25 mM potassium buffer (pH 7.0) for 15 min to lower the pH and then gel was submerged again in a freshly prepared solution containing 18 mM guaiacol and 25 mM H2 O2 in 25 mM K-phosphate buffer (pH, 7.0), till the POD activity-containing band visualized carefully (Fielding and Hall, 1978).

2.3. Measurements of GSH and GSSG 2.6. Statistics For glutathione, 1 g leaves were ground in liquid nitrogen and homogenized in 1 ml 6.67% (w/v) sulfosalicylic acid. Leaf extracts were centrifuged at 20 000 × g for 15 min at 4 ◦ C. Reduced glutathione and oxidized glutathione was determined following Griffith (1980). The total glutathione contents were calculated from a standard curve of reduced GSH. GSH was determined by subtracting GSSG, as GSH equivalents, from the total glutathione content. 2.4. Measurement of lipid peroxidation and LOX activity Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). LOX activity was determined according to Axerold et al. (1981). 2.5. Native PAGE and activity stain Native polyacrlamide gel electrophoresis (PAGE) was performed at 4 ◦ C, 180 V, following Laemmli (1970). For SOD and APX, the enzyme solutions were subjected to native PAGE with 10% polyacrylamide gel and for CAT, the enzyme solution was subjected to native PAGE with a 7% polyacrylamide gel. SDS was omitted from the PAGE. Activity stain for each enzyme was carried out as follows. APX activity was detected by the procedure described by Mittler and Zilinskas (1993). The gel equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM ascorbate for 30 min was incubated in a solution composed of 50 mM sodium phosphate (pH 7.0), 4 mM ascorbate and 2 mM H2 O2 for 20 min. The gel was washed in the buffer for 1 min and submerged in a solution of 50 mM sodium phosphate buffer

Data were subjected to Duncan’s multiple range test using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).

3. Results and discussion A comparison of hyperhydric shoots with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants (Fig. 1A). MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation (Bailly et al., 1996), which is an effect of oxidative damage. This hypothesis is also confirmed by the higher LOX activity in HS plants (Fig. 1B). It indicated that MDA content formation was associated with LOX in leaves that generated more ROS. Generally, LOX activity is observed under wound responses that catalyze the reaction of O2 with free, polyunsaturated fatty acids to form conjugated hydroperoxides. LOX, which is activated in HS, probably plays a role in the elimination of damaged plastids and degradation of chloroplast membrane (Chakrabarty et al., 2005). SOD, POD and CAT activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue (Figs. 2A, 3A and 4A). After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected (Fig. 2B) and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to isoenzymes (1–4). Incubation of gels in 2 mM potassium cyanide or 5 mM H2 O2 before staining for SOD activity

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Fig. 3. (A) Changes in the level of POD activity in the leaf tissues of Euphorbia millii after 30 days of culture (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level). (B) Isoenzyme patterns of POD in hyperhydric E. millii leaves compared with normal in vitro leaves. For POD 150 ␮g proteins were loaded per each well. NH: in vitro grown normal leaf; H: hyperhydric leaf.

Fig. 1. Changes in the level of MDA content (A) and LOX activity (B) in the leaves of E. millii after 30 days of culture. (C) Indicates in vitro grown plantlets. NH: in vitro grown normal leaf; H: hyperhydric leaf (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level).

indicated isozymes SOD-1 to be Mn-SOD, and isozymes SOD-2 to SOD-7 to be Cu/Zn-SOD. One CAT isoenzyme were detected markedly, especially in HS tissue (Fig. 4B). Hyperhydricity also induced single POD isoenzyme activity (Fig. 3B). The oxidative stress is a key component of environmental stress, and increased SOD activity was correlated with increased protection from damage associated with oxidative

Fig. 4. (A) Changes in the level of CAT activity in the leaf tissues of Euphorbia millii after 30 days of culture (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level). (B) Isoenzyme patterns of CAT in hyperhydric E. millii leaves compared with normal in vitro leaves. For CAT 150 ␮g proteins were loaded per each well. NH: in vitro grown normal leaf; H: hyperhydric leaf.

stress (Asada, 1999). Our results showed that the SOD activity in the HS leaves increased significantly. This implies that enhancement of SOD scavenge O2 •− radicals to protect from cellular oxidative damage. The control of the steady-state

Fig. 2. (A) Changes in the level of SOD activity in the leaf tissues of Euphorbia millii after 30 days of culture (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level). (B) Isoenzyme patterns of SOD in hyperhydric E. millii leaves compared with normal in vitro leaves. The different isoforms are numbered from cathode to anode. For SOD 150 ␮g proteins were loaded per each well. NH: in vitro grown normal leaf; H: hyperhydric leaf.


O2 •− levels by SOD is an important protective mechanism against cellular oxidative damage, since O2 •− acts as a precursor of more cytotoxic or highly reactive oxygen derivatives, such as peroxynitrite or HO• (Halliwell and Gutteridge, 1999). Therefore, SOD is usually considered the first line of defense against oxidative stress. Two important sources of oxidative stress in photosynthetic organisms are the chloroplast and the mitochondria. Thylakoids are considered to be one of the major sites of superoxide production because of the simultaneous presence in chloroplasts of a high oxygen level and an electron transport system. Our results proved that the activity of Cu/Zn-SOD constituted the major part of total SOD activity. Chloroplastic Cu/Zn-SOD is attached to the thylakoid membranes (Asada, 1999). However, we do not know which of the different Cu/Zn-SOD isoforms of E. millii is located in the chloroplast. Further studies have to be performed in order to localize SOD isoforms in chloroplasts. In higher plants, Mn-SODs are chiefly present in mitochondria (Halliwell and Gutteridge, 1999; Palma et al., 1986; R´ıo et al., 1992). Interestingly, Mn-SOD activity increased in HS tissue as compared to non-hyperhydric normal plants indicating mitochondria-bound SOD scavenges O2 •− radicals to protect from membrane oxidative damage. From these results it is clear that leakage of electrons to oxygen from electron-transport chains in chloroplasts and mitochondria during photosynthesis and respiration generate H2 O2 in plants (Asada and Takahashi, 1987) and in hyperhydric cells these metabolic activities could be disrupted with the generation of toxic levels of H2 O2 . Therefore, the increase in CAT activity in HS leaves could be necessary to scavenge H2 O2 in peroxisomes and cytosol, where it might have diffused from chloroplasts as a result of SOD activity. Different cell compartments may activate different defensive system to reduce excessive ROS. Similarly total peroxidase activity was considerably higher in leaves of hyperhydric plants. The increased peroxidation of lipids and peroxidase activity that we have observed in our study of hyperhydric leaves could reflect a similar process of oxidative stress with the implication of peroxidase activity as part of the antioxidant response against H2 O2 . However, Franck et al. (2004) argued for a stress response of the HS and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell. The activity of APX was proportionally increased in HS tissue compared to normal leaves (Fig. 5A). After native polyacrylamide gel electrophoresis (PAGE) analysis, one APX isoenzyme was detected markedly, especially in HS tissue (Fig. 5B). APX is primarily located in both the chloroplasts and cytosol, and as the key enzyme of the glutathioneascorbate pathway, it eliminates peroxides by converting ascorbic acid to dehydroascorbate (Izzo et al., 1997); it is one of the most important enzymes playing a crucial role in eliminating toxic H2 O2 from plant cells (Foyer et al., 1994). Therefore, the increased APX activity observed in this study

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Fig. 5. (A) Changes in the level of APX activity in the leaf tissues of Euphorbia millii after 30 days of culture (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level). (B) Isoenzyme patterns of APX in hyperhydric E. millii leaves compared with normal in vitro leaves. For APX 150 ␮g proteins were loaded per each well. NH: in vitro grown normal leaf; H: hyperhydric leaf.

might have been due to the increased H2 O2 production. In the present study, hyperhydricity led to a significant increase in GR, DHAR and MDHAR activity (Fig. 6). The role of GR and glutathione in the H2 O2 scavenging in plant cells has been well established in Halliwell–Asada pathway (Bray et al., 2000). GR is involved in the recycling of reduced glutathione, providing a constant intracellular level of GSH (Calbert and Mannervik, 1985), the main cell antioxidant (Meister, 1981; Alscher, 1989). In the present study, we also investigated enzymes like DHAR and MDHAR related to ascorbate metabolism. The function of MDHAR is to limit the formation of MDHA content through the enzymatic disproportionation, thus generating DHA (Arrigoni, 1994). However, DHA accumulation is harmful to the plant cell (Arrigoni, 1994; Gara et al., 2000). Increased DHAR activity could have generated more ascorbate (AA) from the DHA pool before hydrolysis. Thus, the increased activities of DHAR and MDHAR in HS tissue indicated that these two enzymes catalyzed the regeneration of ascorbate for scavenging of H2 O2 . The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue (Table 1) indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding AA. This AA in addition to the AA produced by non-enzymatic dispropotionation of MDHA was used by APX to directly detoxify H2 O2 . In our study, however, there was a little decrease in GST activity in hyperhydric leaves (Fig. 6). These results suggested that GST is not playing any important role in the detoxification mechanism in hyperhydric leaves, since other enzymes related to antioxidant enzymatic defense system in hyperhydric leaves is highly active. In conclusion, higher LOX and increased lipid peroxidation in hyperhydric E. millii could reflect a similar process of oxidative stress. The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

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Fig. 6. Changes in the level of MDHAR, DHAR, GST and GR activity in the leaf tissues of E. millii after 30 days of culture. NH: in vitro grown normal leaf; H: hyperhydric leaf (different letter within a set of values denotes significant difference by Duncan’s multiple range test at P = 0.05 level). Table 1 Contents of glutathione in leaves of normal in vitro and hyperhydric of E. millii after 30 days of culture Materials

Total glutathione ␮ mol g−1 FW

GSSG ␮ mol g−1 FW

GSH ␮ mol g−1 FW

GSH/GSSG

Hyperhydric Nonhyperhydric

4.289 b 4.8138 a

0.273 b 0.399 a

4.016 b 4.414 a

14.685 a 11.056 b

The same letter within a set of values denotes no significant difference by Duncan’s multiple range test at P = 0.05 level.

Acknowledgements This work was supported by grants from Korea Science and Engineering Foundation (KOSEF) to Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Korea. One of the authors (DC) also wishes to acknowledge the KOSEF for providing financial assistance in the form of “Long-term Foreign Scientist Fellowship”.

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