Ameliorative response of some essential oil furanocoumarins and proteins from Psoralea corylifolia against gamma-irradiation induced oxidative stress

Industrial Crops and Products 76 (2015) 422–431

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Ameliorative response of some essential oil furanocoumarins and proteins from Psoralea corylifolia against gamma-irradiation induced oxidative stress Towseef Mohsin Bhat a,∗ , Sana Choudhary a , M.Y.K. Ansari a , Rumana Aslam a , Sabzar Ahmed Dar b a b

Cell Biology, Molecular Biology and Genetics Section: Department of Botany Aligarh Muslim University, Aligarh 202002, India Centre of Research for Development (CORD), University of Kashmir, Srinagar, J & K, India

a r t i c l e

i n f o

Article history: Received 7 March 2015 Received in revised form 26 June 2015 Accepted 28 June 2015 Keywords: P. corylifolia Gamma radiation Psoralen MALDI-TOF-MS Protein GC–MS

a b s t r a c t Secondary metabolism producing essential oil furanocoumarins depends on various environmental factors and ionizing radiation is currently a very important way of enhancing these compounds through radio stimulation. The study aims to evaluate the ameliorative effect of essential oil furanocoumarins and proteins from Psoralea corylifolia against gamma-irradiation-induced oxidative stress. Antioxidant biomarkers like H2 O2 , peroxidase, superoxide dismutase, and catalase increased significantly (p < 0.01) in all treated groups. Dose dependent increase of H2 O2 concentration leads to significant chromosomal abnormalities. Essential oil (EO) yield and furanocoumarin content showed a significant (p < 0.01) increase up to 200 Gy. These contents decreased significantly on increased expression of proteins involved in furanocoumarin biosynthesis and stress defense. DPHH and Phosphomolybdenum assays showed a significant (p < 0.01) antioxidant activity in the essential oil on varying doses of gamma radiation. It is concluded that mild doses of gamma irradiation is an important abiotic-elicitor for enhancing the essential oil yield and furanocoumarin content in P. corylifolia which possess important radio resistant properties. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Linear furanocoumarins, particularly psoralen synthesized in the seeds of Psoralea corylifolia, possesses immense biomedical applications against various diseases. They are particularly promising candidates against oxidative damage induced pathological and degenerative processes recommended for the treatment of skin diseases including aging and cancer and also in the treatment of demyelinating symptoms, (Baskin et al., 1967; Ekiert and Czygan, 2005). Biological properties of furanocoumarins and furoquinolone alkaloids makes them fascinating in pharmaceutical industry, hence substantial interest has been shown for their availability and sources. Commercial production of these secondary metabolites is usually restricted by their low yield. Plants produce furanocoumarins mainly in reverberation to biotic or abiotic insults such as irradiation, infections, wounding and exposure to ozone,

∗ Corresponding author. E-mail address: [email protected] (T.M. Bhat). http://dx.doi.org/10.1016/j.indcrop.2015.06.059 0926-6690/© 2015 Elsevier B.V. All rights reserved.

pollutants, and other harsh environmental conditions (Korkina et al., 2011). Molecular mechanism behind protective nature of phenylpropanoids in plants reflects in their important antioxidant and free radical scavenging properties (Korkina, 2007). Mechanisms of action of biotic and abiotic elicitors have been differently viewed; they are complex, and many hypotheses have been put forward in finding the proper mechanisms of action. Very limited information is available regarding the biosynthetic routes of secondary metabolites and what role does an elicitor/stressor plays on a plant cell or tissue culture is very difficult to predict. Major elicitation approaches and studies are therefore empirical (Shibuya and Minami, 2001). The operational mechanism of elicitors is one of the most important studies to deal with the necessary biotechnological systems for producing these metabolites. Furanocoumarins belong to an important class of phenolic secondary metabolites which is sub-grouped among more than 1500 coumarin compounds involved in important defense mechanisms against pathogens, insects, and as well as to reduce the stress in plants (Bourgaud et al., 2006). They are mostly found in higher quantities in four families viz. Apiaceae, Rutaceae, Fabaceae

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and Moraceae. They take an active role by fighting against phytopathogens/stressors or insects (Ojala et al., 2000), and may also cause inter-specific competition by suppressing the germination, growth and development of neighboring plants (Baskin et al., 1967). In Babchii (P. corylifolia), and other fabaceae plants, these compounds primarily accumulate in oil ducts (Fisher and Trama 1979; Zobel and Brown, 1990). Two distinct pathways for furanocoumarin biosynthesis has been derived one among them is linear pathway for the synthesis of psoralen derivatives and another one is for the synthesis of angelicin derivatives. It has been hypothesized that these two pathways originated as a result of general co-evolution between insects and plants. Many enzymes involved in furanocoumarin biosynthesis have been described during recent years at molecular level which include two P450 enzymes (psoralen synthase and angelicin synthase; Larbat et al., 2009), bergaptol O-methyltransferase (Hehmann et al., 2004), and a Fe/oxoglutarate dependent dioxygenase of 4-coumaroyl CoA (C2 ’H) that yields umbelliferone, a central intermediate of coumarin derivatives (Vialart et al., 2012). P. corylifolia produces numerous flavonoids, among them aurones, flavons, flavonols, anthocyanins, condensed tannins, flavonones and isoflavones are synthesized through phenylpropanoid pathway. These compounds are used in pharmaceutical industry for the protection against cardiovascular diseases, hormone dependant cancers, menopausal symptoms and osteoporosis (Cornwell et al., 2004; Misra et al., 2010). These flavonoids have also been used in plant-microbe/fungal interactions and their synthesis is induced by defense signal elicitors, such as jasmonic acid and salicylic acid (Misra et al., 2010). The formation of isoflavonoids is activated by a legume-specific cytochrome P450 enzyme, 2-hydroxyisoflavanone synthase commonly known as IFS (Misra et al., 2010; Parast et al., 2011). The IFS activated reaction byproducted into the generation of 2-hydroxyisoflavanone intermediate which is inconsistent and dehydrates to generate the corresponding isoflavone either automatically or with the help of an enzyme hydroxyl isoflavanone dehydratase (Akashi et al., 2005). IFS gene has been cloned from Glycine max, Trifolium and red clover which belong to a multigene family (Jung et al., 2000; Shimada et al., 2000). Several workers observed the gamma radiation induced elicitation pathway for the augmentation of furanocoumarins; Psoralen content in P. corylifolia by (Ahmed and Baig, 2014; Jan et al., 2012); Umbelliferone content in Ruta graveolens by (Vialart et al., 2012), furanocoumarin content in Ruta graveolens by (Karamat et al., 2012, 2014) etc. Many studies have been performed by different workers on the dose-response effects of ionizing radiation, particularly gamma radiation on various growth and yield related characters in plants. Induction of chromosomal alterations after gamma ray treatment induce stress response due to the over production of reactive oxygen species (ROS) and accumulation of the psoralen which is an antioxidant and acts as a switch to overcome the stress (Bhat et al., 2015). There occurs an enhanced protein expression related to stress and most of such proteins have been studied earlier by workers (Hachez et al., 2014; Bhat et al., 2015). Present work was undertaken to study the dose dependent response of gamma rays as an abiotic elicitor/stressor for the enhancement of some furanocoumarins especially psoralen from the essential oil using GC–MS–FID. If the irradiation is the direct cause of metabolite profile changes, chromosomal and antioxidant activities were undertaken. Proteome analysis is a powerful approach in the field of functional genomics to identify the function of the proteins/genes driving the particular reaction. In order to show biochemically that which proteins are responsible for increased psoralen biosynthesis and for ROS scavenging, 2D-potein profiling with MALDI-MS analysis was carried out to identify the proteins involved in the pathway.

423

2. Experimental 2.1. Germplasm procurement and Irradiation Certified healthy seeds of P. corylifolia (Var: Pratap Babchi-1) were obtained from National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Respective sample (25 g) of seeds was packed in polythene bags and irradiated with varying doses of gamma rays (50, 100, 150, 200 and 250 Gy at room temperature 28 ±2 ◦ C)60 Co (Gamma chamber, GC. 9000 at National Botanical Research Institute, Lucknow, India). The non-irradiated set of seeds was taken as control. The control and the treated sets of seeds were sown in sandy loam type of soil having pH 7.4, total nitrogen 0.08(%), total phosphorus 0.66(%), and organic carbon 1.91(%) in the agricultural field of Aligarh Muslim University Aligarh, India in the month of March-2013. Each sample was planted in six rows, 40 cm apart. Care was taken to avoid root disturbances as this plant is more sensitive to root disturbances. Agricultural practices like irrigation and weeding were carried out whenever required. 2.2. Measurement of H2 O2 H2 O2 content was determined according to the method of Okuda et al., (1991) with minor modifications as suggested by Asgher et al., (2014). Ten-days old seedlings (1 g of fresh weight (FW) were homogenized in 5 ml of cold acetone, and centrifugation was carried out to the homogenate at 5000 g for 30 min. Collected supernatant was added into a concentrated hydrochloric acid solution of 0.1 ml 20% TiCl4 and 0.2 ml concentrated ammonia. After five minutes at 25 ◦ C the reaction mixture was centrifuged at 16,000 g and 4 ◦ C for 20 min. Cold acetone twice was used for the washing of pellets and then mixed with 2 ml 1 M H2 SO4 . Absorption was measured at 410 nm and the concentration of H2 O2 was determined from a standard curve plotted with known concentrations of H2 O2 control. 2.3. Chromosomal aberrations For chromosomal aberration studies, young flower buds from each dosage level and from the control were fixed in freshly prepared Carnoy’s fixative (absolute alcohol, chloroform, and acetic acid in a ratio of 6:3:1 v/v) for 24 h and kept in 70% alcohol for further use. Collected anthers were squashed in 1.5% propionocarmine and made permanent through an alcohol series following the method of Sax, (1939). Photographs were taken from both temporary and permanent slides using a high resolution (Dsx 100 Olympus) Microscope. 2.4. Harvesting and drying All samples were obtained on August-2014. P. corylifolia was at flowering stage at the time of harvest, to ensure best essential oil content and composition. P. corylifolia plants were harvested from each dose of gamma radiation and each harvest was taken in 6 replicates, resulting in 36 P. corylifolia samples. The fresh psoralea samples for oil content and composition were dried in a well-ventilated room at shade. 2.5. Essential oil extraction using Clevenger All P. corylifolia biomass samples for oil content and composition were extracted through steam distillation in 2-L steam distillation units for 60 min by the method of Zheljazkov et al., (2010). The beginning of each distillation was measured when the first drop of essential oil was out of the condenser and in the separator. At

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Table 1 Chromosomal lesions studied at different meiotic stages in P. corylifolia irradiated with different doses of gamma radiations. Gamma rays

Control 50Gy 100Gy 150Gy 200Gy 250Gy

PMCs

123 13 125 127 131 129

Metaphase-I/II

Anaphase-I/II

Telophase-I/II

% of total ab. PMCs in all stages

PM

Sc

% of ab. cells

L

B

% of ab. cells

L

B

M

% of ab. cells

– 2 2 3 4 6

– 1 2 2 3 4

– 2.20 ± .19** 3.30 ± .25** 3.94 ± .31** 5.34 ± .43** 7.75 ± .63**

– 1 2 2 3 4

– – 1 2 2 1

– 0.73 ± .08** 2.48 ± .19** 3.15 ± .25** 3.82 ± .31** 3.87 ± .36**

– 1 1 2 3 5

– 1 1 2 3 4

– – 1 1 2 3

– 1.47 ± .12** 2.48 ± .15** 3.94 ± .26** 6.10 ± .40** 9.30 ± .61**

– 4.41 ± .24** 8.26 ± .35** 11.02 ± .47** 15.26 ± .68** 20.92 ± .94**

PMCs: Total number of PMCs observed, PM, precocious movement; Sc, stray chromosomes; ab. cells, abnormal cells; L, laggards; B, bridges, M, multinucleate. Results are expressed as mean ± SD (n = 12). Values with different superscripts differ significantly (* P < 0.05: significant, ** P < 0.01: highly significant, *** P < 0.001: extremely significant) from the control when analyzed by one-way analysis of variance (Newman–Keuls and Dunnett’s multiple comparison test).

Table 2 Mean oil yield (oil content g/100 g dry weight, adjusted to 60% moisture), the concentration (%) and yield (mg/g dried material) of Psoralen, Bakuchiol and ␤-caryophyllene measured after gamma irradiation treatment in P. corylifolia L. Value represents mean ± SE (n = 6). Doses

Oil yield (%)

Psoralenconc. (%)

Bakuchiol conc. (%)

␤-caryophyllene conc. (%)

Psoralen conc. yield (mg/g)

Bakuchiol yield (mg/g)

␤-caryophyllene yield (mg/g)

Control 50Gy 100Gy 150Gy 200Gy 250Gy

0.90 ± 0.01 0.90 ± 0.01 0.91 ± 0.01 0.92 ± 0.02a 0.93 ± 0.02a 0.92 ± 0.01

14.80 ± 0.14 27.76 ± 0.04b 32.33 ± 0.05b 34.21 ± 0.06b 35.43 ± 0.04b 34.23 ± 0.04b

12.30 ± 0.02 14.44 ± 0.04b 15.11 ± 0.00b 20.44 ± 0.04b 22.36 ± 0.01b 22.22 ± 0.01b

8.50 ± 0.03 8.84 ± 0.02b 8.98 ± 0.01b 9.34 ± 0.04b 9.85 ± 0.02b 9.81 ± 0.02b

22.99 ± 0.52 24.61 ± 1.29 33.56 ± 1.09b 36.33 ± 0.48b 44.76 ± 2.38b 43.16 ± 1.52b

18.55 ± 1.33 21.39 ± 1.41b 24.95 ± 0.09b 26.28 ± 1.18b 26.70 ± 2.22b 25.99 ± 0.94b

17.49 ± 1.49 19.02 ± 0.44 19.44 ± 1.82 22.31 ± 4.63b 24.91 ± 0.89b 23.67 ± 0.61b

Values with different lower alphabet superscript differ significantly (a p < 0.05: significant. b p < 0.01: highly significant. c p < 0.001: extremely significant) between control and treated groups (Bonferroni’s test).

the end of each distillation, the power was turned off; the oil and the water were decanted from the separator into glass vials. The oil was separated from the water, the oil weight measured on an analytical scale, and kept in a freezer at 16 ◦ C until the analyses. The essential oil content (yield) was calculated as grams of oil per 100 g of fresh herbage after corrected for moisture content, using the difference between the fresh and dried weight of the biomass samples.

2.7. Assessment of Antioxidant activity of essential oil The radical scavenging activity of the essential oil extract was determined DPPH (2, 2-diphenyl-1-pycrilhydrazil hydrate) and phosphomolybdenum reduction assay following (Shinde et al., 2010) with minor modifications (Amessis-Ouchemoukh et al., 2014). The free radical (DPPH) scavenging activity was expressed in terms of % inhibition whereas phosphomolybdenum reduction capacity of extract as equivalents of ascorbic acid (␮mol/g of extract) at 50 ␮g/ml concentrations.

2.6. Gas chromatography-FID-quantification 2.8. Proteomic analysis of Seeds All P. corylifolia oil samples were examined using GC–MS (Shimadzu QP-2010) fitted with FID and a QP-5000 (Quadrapole) mass spectrophotometer. A fused silica capillary column DB5(30 m × 0.25 mm, with a film thickness of 0.25 ␮m) was conducted using the conditions like injector temperature 240 ◦ C; column temperature 60–100 at 3 ◦ C/min, kept at 240 ◦ C at20 ◦ C/min for 2 min, carrier gas Helium; injection volume, 1 ␮L (spliton FID, split ratio 20:1); FID temperature was 250 ◦ C. Analytical grade Psoralen (Sigma–Aldrich), ␤-caryophyllene (Sigma–Aldrich) and bakuchiol (Sigma–Aldrich) were used for quantitative analysis of P. corylifolia oil constituents. Quantitative analysis of essential oil was already described by Zheljazkov et al., (2010) and is concisely described. Every standard was used to create a separate calibration curve using FID response data. Regression coefficients and response factors (RF) was imposed for linearity independently. Equation = DR/C were used for Response factor(RF) calculation where DR was the detector response in peak area(PA) and C is the concentration of the analyate. The chromatograms of each of the essential oil samples from all doses including control and replicates were compared to the chromatograms from standards. Target peaks were confirmed by retention time. Percentage of each chemical constituent in the essential oil was confirmed from integrated peaks. In order to determine the percentage of that constituent in each essential oil sample by equation (PA/RF/C) × 100 = % (peak area/response factor/concentration) the RF of the target chemical constituent was used.

Proteins were isolated from the embryonic axis of irradiated seeds as well as from the control after harvesting to determine the radiation induced changes due to gamma rays. Seeds were ground into powder form using liquid nitrogen and 400 mg of the P. corylifolia seed powder was weighed and kept in a polypropylene tube with 4 ml of a solution having 40% of isopropanol. Protein extraction was done using an orbital shaker (Sorvell Instrument, Dupont) for one and a half hour at 17,000 rpm. Centrifugation was carried out at 12,000 rpm for 8 min at 4 ◦ C. Supernatant was separated and kept in a clean 10-ml tube by adding10 ml of cold acetone and then vortexed thoroughly. Further incubation was done to the extract at −20 ◦ C for 12 h, after that samples were centrifuged for 15 min at 7000 rpm at 4 ◦ C and the pellet was dried for 25 min and then resuspended in 0.4 ml of lysis buffer [9 M urea, 1% CHAPS, 0.5% IPG buffer (pH 4–7) and 1% DTT].Then quantity of the protein was determined by 2D-PAGE analysis following the methods of Natarajan, (2002) using a commercial dye reagent (Bio-Rad). 2.8.1. 2D Gel electrophoresis Isoelectric focussing was carried out using 13 cm, pH 4–7, linear IPG strips in the EttanIPGphor 3 IEF System (GE Healthcare). All IPG strips were rehydrated with 230 ␮l rehydration buffer (9.8 M urea, 4% CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue), containing 100 ␮g of protein and DTT. Isoelectric focussing was done at 500 V for 1 h, 1000 V for 1 h and 8000 V. Immediately the focused

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Fig. 1. Effect of different doses of gamma radiation on H2 O2 production and antioxidant enzyme production of P. corylifolia (a) H2 O2 concentration (b) POD content (C)SOD content (D) CAT content. Values with different lower alphabet superscript differ significantly (a p < 0.05: significant. b p < 0.01: highly significant. c p < 0.001: extremely significant) between control and treated groups.

strips were run on a second-dimension gel electrophoresis. The gel strips were incubated with equilibration buffer 1 (3.0 M Tris–HCl, pH 8.0, 6 M urea, 30% glycerol, 0.4% SDS, 0.002% bromophenol blue, 1% DTT) and equilibration buffer 2 3.5 M Tris–HCl, pH 8.0, 6 M urea, 30% glycerol, 2% SDS, 0.003% bromophenol blue, 2 % iodoacetamide) for 15 min each on a shaker.After removing the equilibration buffer, the strips were washed with water and kept on 12% polyacrylamide gel (8 × 13 cm) with Tris-glycine buffer system. The 2D-PAGE gels were visualized by staining with Coomassie Brilliant Blue G-250 (colloidal). The gels were fixed overnight in 50% ethanol and 10% acetic acid followed by 2 × 20 min washes with deionized water. Then the gels were pre-stained for 1 h in 34% methanol, 17% ammonium sulfate, and 3% phosphoric acid, and then stained in the same solution. Laser densitometry (PDSI, GE Healthcare) and Image Master 2D-Elite (version 4.01) software were used for the analysis of the gels. 2.8.2. In-gel digestion of protein spots Protein spots of interest were blotted out carefully from the 2D gels, with each spot in a separate micro-tube, and protein digestion was performed following the methods of Qureshi et al., (2010). The excised gel pieces containing protein were washed with CH3 CN:H2 O (1:1, v/v), containing 25 mM ammonium bicarbonate to extripate the blue stain. The gel pieces were dehydrated with 100 % acetonitrile, dried and incubated overnight at 37 ◦ C with 20 ␮l of 10 ␮g/ml porcine trypsin prepared in 20 mM ammonium bicarbonate. Tryptic pieces were eluted by diffusion into CH3 CN:H2 O and 0.5% trifluoroacetic acid (1:1, v/v). The extract was vacuum-dried and the pellet was dissolved in CH3 CN:H2 O and 0.1 % trifluoroacetic acid (1:1, v/v).

2.8.3. Mass spectrometry Voyager DESTR MALDI-TOF mass spectrometer (Model 4800, Applied Biosystems, UK) was used for peptide mass fingerprinting (PMF), operating in positive ion reflector mode was used to analyze the tryptic peptides. Co-crystallization of the samples were carried out with cyanohydroxycinnamic acid (CHCA) matrix, and spectra were acquired with 400 shots of a 337 nm nitrogen laser operating at 20 Hz. The ‘merge pl’ script from Matrix Science was used to convert multiple Sequest DTA files into a single Mascot generic file suitable for searching in Mascot.

2.8.4. PMF data analysis Protein identification was carried outusing the Mascot search engine (http://www.matrixscience.com), which uses a probabilitybased scoring system (Cottrell and London, 1999). NCBI nonredundant and Swiss prot databases were selected as the primary databases to be searched for protein sequence matches.

2.9. Statistical analysis A total of 5 replicates for each treatment were conducted. Statistical analysis of data was done with SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). Data was compared for statistically significant differences between control and treatment groups using one-way analysis of variance (ANOVA). Significant differences in ANOVA were further analyzed by DMRT with Bonferroni’s / Tukey tests.

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Fig. 2. Chromosomal alterations studied at various stages of meiotic cell cycle in P. corylifolia irradiated with varying doses of gamma radiations. (A) Laggard at metaphase (B) Laggard at anaphase (C) Precocious movement (D) Bridges (E) Stray chromosome (F) Micro nuclei.

3. Results and discussion 3.1. Effects of gamma irradiation on ROS level and antioxidant enzyme activities Reactive oxygen species was observed by determination of hydrogen peroxide (H2 O2 ) in P. corylifolia leaves exposed to varying doses of gamma irradiation (Fig. 1a). The concentration of H2 O2 was observed highest by more than 33% respectively under high dose (250 Gy) of gamma radiation as compared to control. In plant cells, ROS are obligatory side products of aerobic metabolism. The amount of ROS is modest under normal growth conditions, and cells experience only exquiste oxidative stress, whereas, many abiotic stresses like gamma radiations increases ROS production (Jan et al.,2012; Golari et al., 2014). The results of our study clearly shows that gamma irradiation triggered the rapid induction of H2 O2 generation in P. corylifolia seedlings. This enhanced production of ROS under stress can pose a threat to cells and can also act as a signal to activate stress response pathways involving hyper accumulation of furanocoumarins (Abdeldaiem and Hoda, 2014; Jan et al., 2012). As a response to ROS overproduction, plant cells activate endogenous antioxidant enzyme defensive activities (Gill et al., 2013). As shown in (Fig.1b–d) the POD, SOD and CAT activity increased significantly (p < 0.01) up to 62%, 56% and 45% respectively as compared with the level observed in the control. Further

CAT exhibited higher activities than SOD in both the irradiated and non irradiated progenies (Fig. 1b–d). Oxidative stress is induced by gamma irradiation with overproduction of reactive oxygen species (ROS) such as hydrogen peroxides (H2 O2 ) which react expeditiously with almost all structural and functional organic molecules, including proteins, lipids and nucleic acids causing cellular metabolism disturbances, this finding suggest that H2 O2 is the central free radical of the ROS induced by gamma rays. Our results are concomitant with previous results of many workers (Jan et al., 2012; Qi et al., 2015). Greater amount of SOD and APX activities was found in peppers exposed to low doses of gamma irradiation (Kim et al., 2005). Recent finding of Kim et al., (2011) reported that Arabidopsis plants on exposure to 100 and 800 Gy gamma radiation increased the level of SOD at vegetative stage and that CAT activities are differentially regulated depending on the stage of the plant and irradiation doses (Pérez et al., 2007). ROS metabolism in plant cells is under the control of antioxidant enzymes, our results depict that enhanced activities of POD, SOD, and CAT are the best antioxidant scavengers under gamma irradiation stress and add to the maintenance of the relatively non toxic level of ROS in cells (Macovei et al., 2014). Reactive oxygen species (ROS) have now become a buzz word in recent years in stress biology because plants surfeited with mechanisms to combat it by involving various biological processes such as growth and development, programmed cell death (Apel and Hirt, 2004). Several workers viewed that induced ROS level by low-

Table 3 List of protein from two-dimensional gel electrophoresis (2-DGE) identified by mass spectrometry (MS). Spot. No.

Protein species

Gene bank accession number

Protein pI/Mw

% coverage

Peptidecount

Species

E-Value

Sub cellular location

Identity (%)

Expression

1.

1. Stress defense Cu/Zn superoxide dismutase

AAD42179.1

4.80/24

81

82

Vigna radiata

9e-16

cytoplasm

42

2.

Glutathione synthetase precursor

AAF98121.1

4.60/17

87

99

Pisum sativum

0.0

chloroplast

70

3.

Homoglutathione synthetase

AES82542.2

4.35/37

87

105

Medicago trunculata

0.0

chloroplast

71

up regulated C, 100Gy, 200Gy, 250Gy up regulated C, 100Gy, 200Gy, 250Gy up regulated C, 100Gy, 200Gy, 250Gy down regulated C, 150Gy up regulated C, 100Gy, 200Gy, 250Gy down regulated C, 150Gy

Chalcone synthase

XP003624524.1

4.25/12

98

58

Medicago trunculata

0.0

cytoplasm

85

Isoflavone reductase like protein

XP006581203.1

4.27/23

81

112

Glycine max

9e-95

chloroplast

81

6.

Isoflavone reductase

CAA4110.61

6.25/8

100

67

Medicago sativa

2e-78

7.

2. Metabolism of carbohydrates Enolase

NP001237329.1

4.65/23

99

88

Glycine max

0.0

chloroplast

89

8.

Phosphoglycolate phosphatise

XP004492869.1

4.40/11

86

54

Cicer arietinum

4e-80

chloroplast

76

9.

4nitrophenylphosphatase protein

BAB11323.1

4.35/55

81

64

Arabidopsis thaliana

1e-74

chloroplast

67

10.

Fructosebisphosphate aldolase

NP001237315.1

4.50/5434

100

25

Glycine max

0.0

cytoplasm

59

11.

Ribulose-1,5 bisphosphate carboxylase

CAJ86884.1

5.95/45

7

45

Psoralea aculeata

2.4

chloroplast

14

12.

3. Furano coumarin production Isoflavone synthase

ACA81476.1

6.05/35

100

54

Glycine soga

6e-154

chloroplast

93

13.

Isoflavone synthase 2

AAF34534.1

6.60/12

95

65

Lupinus albus

3e-139

chloroplast

91

14.

Geraniol-8- hydroxylase

XP004495721.1

6.65/43

92

88

Cicer aeritinum

1e-97

cytoplasm

30

15

RNA polymerase beta subunit

AFK10007.1

4.80/23

65

83

Cullen corylifolium

5e-20

chloroplast

91

16

Cytochrome oxidase sub unit

AAF15336

6.70/36

27

76

Psoralidum lanceolatum

1e-08

cytoplasm

13

17

Isoflavone synthase

ADB93869.1

4.27/47

65

45

Cullen corylifolium

3e-04

chloroplast

82

18

Enolase like

XP003548246

4.50/25

88

100

Glycine max

1e-21

mitochondria

43

19

Uncharacterized protein LOC

NP001238239.1

6.75/46

56

71

Glycine max

2e-13

cytoplasm

22

20

Cytochrome oxidase sub unit

AAF15336

6.70/36

27

76

Psoralidum lanceolatum

1e-08

cytoplasm

13

57

up regulated 50Gy, 100Gy, 150Gy, 200Gy, 250Gy down regulated C up regulated 50Gy, 100Gy, 150Gy, 200Gy, 250Gy down regulated C up regulated 5Gy, 100Gy, 150Gy, 200Gy, 250Gy down regulated C up regulated 5Gy, 100Gy, 150Gy, 200Gy, 250Gy down regulated C up regulated 5Gy, 100Gy, 150Gy, 200Gy, 250Gy down regulated C up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy,150Gy, 200Gy,250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy up regulated 100Gy, 150Gy, 200Gy, 250Gy down regulated C, 50Gy

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4. 5.

427

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Fig. 3. Antioxidant activity of P. corylifolia essential oil upon treatment with varying doses of gamma radiation. (a). DPPH assay EC50 value (␮g/ml) (b). Phosphomolybdenum assay (50 ␮g/ml). Values with different lower alphabet superscript differ significantly (a p < 0.05: significant. b p < 0.01: highly significant. c p < 0.001: extremely significant) between control and treated groups. The values with different upper alphabet superscript differ significantly (A p < 0.05: significant. B p < 0.01: highly significant. C p < 0.001: extremely significant) between 50 Gy group with other treated groups, whereas values with different numeric superscripts differ significantly (1 p < 0.05: significant. 2 p < 0.01: highly significant. 3 p < 0.001: extremely significant) between 100 Gy group and other treated groups (Dunnett’s multiple comparison test).

dose gamma irradiation functions as signal in the radiation-induced hormetic effects in plants (Kim et al., 2004, 2005). 3.2. Effects of gamma irradiation on chromosomes In order to confirm the target principle of ionizing radiation, chromosomal studies was much reliable way to confirm that DNA damage or genotoxic events as a direct consequence of the exposure of biological macromolecules to charged particles inducing ROS signal. Chromosomes were normal in control plants showing 11 bivalents in diakinesis, however, a number of chromosomal aberrations were observed in the plants raised from the seeds irradiated with different doses of gamma radiation. Spectrum of chromosomal aberrations due to gamma radiation at various stages of cell division cycle is depicted in (Fig. 2, Table 1). Most frequent aberrations depicted in meiotic phase of cell cycle showed stray chromosomes, laggards, bridges and micronuclei. All the chromosomal aberrations increased with increase in gamma radiation dose from 50 Gy to 250 Gy. Chromosomal alterations like precocious movement and stray chromosomes, studied at metaphase I and II of cell cycle, showed significant induction (2.20 to 7.75%; p < 0.01) on increasing in the gamma radiation dose. Laggards, bridges and multinuclei observed in anaphase and telophase I/II also showed a significant increase (p < 0.01) at higher doses. Quantitative relationship between a dose of radiation and the yield of aberrations depends on both the type of aberration and the kind of radiation. In gamma irradiated plants simple chromosomal

aberrations are linearly related to the dose. Chromosomal aberrations during gamma radiation exposure occur mainly as a result of production of ROS (Qi et al., 2015). Some general theory of aberration production by ionising radiation, based on the previous studies (Giles, 1954; Natrajan, 2002; Jagetia et al., 2003), states that a break in either single or divided chromosome is due to direct action on it by the ionisation produced by the reactive species like an electron, proton or ␣-particle. Such a break can remain as such to give rise to terminal deletion, rejoin in the original position or join with an adjacent break in the same or different chromosome to produce various types of aberrations. 3.3. Phytochemical screening The major compounds psoralen, Bakuchiol and ␤-caryophyllene were identified and quantified in the essential oil of P. corylifolia. The oil yield (content) showed a significant (p < 0.05) increase in the treated populations as compared to the control and the maximum yield at 200 Gy (Table 2). Concentration of psoralen, Bakuchiol and ␤-caryophyllene also showed a significant (p < 0.01) increase in the treated groups with respect to control. Maximum essential oil (0.93%; p < 0.05); psoralen (35.43%; p < 0.01), bakuchiol (22.36%; p < 0.01), and ␤-caryophyllene (9.85%; p < 0.01) was observed at 200 Gy (Table 2). Gamma radiations () enhance the production of different plant metabolites (Kim et al., 2005). Stimulation of secondary metabolites due to UV light reported earlier include phenolic compounds,

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Fig. 4. 2D gel analysis of proteins extracted from seeds irradiated with different doses of gamma rays. In the first dimension (IEF), 150 ␮g of protein was loaded on a 17-cm IPG strip with a linear gradient of pH 4–7. In the second dimension, 12% SDS-PAGE gels were used. Proteins were visualized by silver staining. The rounds indicate those proteins which are shown differential expression.

surface flavonols and flavonoids (Harborne and Williams, 2000), anthocyanins (Oelmüller and Mohr, 1985), etc., and these compounds have been implicated in plant’s defense processes (Chappell and Hahlbrock, 1984) and protection against UV-light (Ziska et al., 1992). It is reported that blue light enhanced anthocyanin production in straw berry cells, whereas red light hardly affected them (Kurata et al., 2000). Based on a previously published mathematical model analyzed with hormetic-like data, Fornalski and ´ (2012) proposed that hormesis is typically observed at Dobrzynski, doses less than 30 Gy, and that no observed adverse effect level point may be located between 30 and 100 Gy for the irradiated ´ 2012). These findings are not conseeds (Fornalski and Dobrzynski, sistent with our results, because an obvious inhibitory effect was detected at high doses (250 Gy) of gamma rays. Several studies have explained the stimulatory effects of low-dose gamma radiation on furanocoumarin content (Zobel et al., 1993; Jan et al., 2012). The exposure of plant tissues to low-dose gamma radiation was found to induce changes in the primary metabolism of the plant similar to biotic and abiotic stimuli. Significant changes in the relative abundance of multiple metabolites were observed by various workers and are the result of genetic reprogramming of primary metabolism in response to stress (Broeckling et al., 2005; Farag et al., 2008).

3.4. Antioxidant activities of essential oil The most effective DPPH radical scavenging and phosphomolybdenum reducing property of essential oil derived from irradiated and non irradiated plants showed a significant (p < 0.01) increase in the antioxidant activity in the treated groups with respect to control (Figure 3a and b). The highest increase in DPHH and phosphomolybedinium activity by 48% and 20% respectively was observed

in plants irradiated with 200 Gy gamma dose. At 250 Gy both the activities showed a significant (p < 0.01) decrease as compared to the mild doses but the activity was still higher than control. There was a significant variation in the antioxidant activities in between the treated groups. Antioxidant activity had remained an earth shattering word and the subject of rigorous investigations due to the ever-increasing demand by the food and pharmaceutical industries for developing natural bioactive antiaging and anti-carcinogenic compounds that demonstrate measurable health benefits (Choi et al., 2009). There are very few studies in the literature on an antioxidant activity associated with essential oil of medicinal plants. In this study, we investigated the changes of antioxidant activities with two different methods. Fig. 3(a) shows the DPPH radical scavenging activity of essential oil treated by the gamma irradiation. DPPH radical scavenging activity showed a significant increase because of increased psoralen content. Antioxidant modulation of psoralen by gamma radiation has been observed by various workers like (Baskin et al., 1987; Jan et al., 2011, 2012; Siva et al., 2014). The DPPH radical scavenging activity was increased continuously with increasing dose up to 200 Gy. In Fig. 3(b), the change of the phosphomolybidinium activity of essential oil is also shown. There was a remarkable increase in the scavenging activity in the treated plants as compared to control. The increase in the antioxidant activities of the essential oil of P. corylifolia was concomitant with the earlier studies. Variyar et al., (2004) reported that DPPH radical scavenging activity of soybean was enhanced up on gamma irradiation dose. Ahn and Nam, (2004) reported that, after irradiation, a scavenging ability of phytic acid was more than non-irradiated. Kim et al., (2008) reported that the hyaluronic acid showed the increased DPPH radical scavenging activity by the gamma irra-

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diation. There are no conclusive explanations for the enhanced antioxidant activity following low dose gamma radiations. The increase in antioxidant activity up to 200 Gy dose may be attributed due to photoactivation of some enzymes of the terpenoid pathway through which essential oil is produced in plants. Photoactivation of the enzymes for psoralen and isopsoralen production proved to enhance the antioxidant activity (Hamerski et al., 1990; Ali et al., 2011).

tein expression to cope the radiostress at 250 Gy.This study finds its importance in enhancing the furanocoumarins having phytoactive medicinal values and antioxidant properties to combat radiological stress. Besides, furanocoumarins have also a broad range of pharmacological activities such as photosensitizing, photobiological and phototherapeutic properties to play in pharmacological industry. Acknowledgement

3.5. Genomic alterations using 2D-gel electrophoresis Variability depicted through two dimensional gel electrophoresis resulted in the identification of 100 protein spots. Among them, 20 proteins showed differential expression. Out of 20 differentially expressed proteins, six (06) proteins were found to exhibit similar patterns of differential expression in both control and irradiated populations. Out of these six proteins, four proteins (spots 1, 2, 3 and 5) were found to be up-regulated in control, 100 Gy, 200 Gy and 250 Gy and two proteins (spots 4 and 6) were found to be down regulated in control, 150 Gy dose of gamma radiation. About 5 proteins of the second class (spots 7, 8, 9, 10 and 11) involved in metabolism of carbohydrates were found to be up-regulated in every dose of gamma radiations but not in the control. Third class of nine different proteins (spots 12, 13, 14, 15, 16, 17, 18,19 and 20) involved in production of secondary metabolites were found to exhibit increased patterns of differential expression in the treated populations when compared to the non irradiated control (Table 3, Fig. 4). All these nine protein were found to be up regulated in all doses except 50 Gy. Conventional two-dimensional gel electrophoresis (2-DE) approach coupled with protein identification by mass spectrometry (MS) has been the most extensively used proteomic technique for investigation of gamma radiation induced alteration of plant proteome composition. A high degree of variability have been observed in the expression of P. corylifolia seed proteins subjected to varying doses of gamma radiations involved in stress defense, photosynthesis and secondary metabolite production (Table 3, Fig. 5). Modulation of plant proteome composition is an important process to cope with the environmental insults including radiological stress. P. corylifolia has evolved complex mechanisms to cope with the alleviated stress damages. Functional translated portion of the genome plays an important role in plant stress response, proteomic studies provide us a finer picture of protein networks and metabolic pathways primarily involved in radioprotection (Romain et al., 2009; Alves et al., 2011; Barcelos et al., 2011). Accumulation of proteins is one of such plant defense strategies and often associated with radio stress (Soheila, 2000). 4. Conclusion The present work examined phytochemical response of P. corylifolia against varying doses of gamma rays and the role of psoralen and defensive proteins to cope the radiological stress. Lower doses of gamma rays provide modulatory role in enhancing the growth and developmental processes while higher doses of gamma rays produced a severe effect on almost all variables. This effect was greater in the seeds treated with 250 Gy of gamma rays. Radiation induced chromosomal abnormalities increased at higher doses of gamma rays leading to an important radiobiological response which at cellular level can alter the antioxidant machinery and protein regulation in P. corylifolia. With all these examples, it is concluded that mild doses of gamma radiation are beneficial for the improvement of essential oil yield and other antioxidant activities in this experimental crop. Essential oil and furanocoumarin content improved significantly over control in moderate doses up to 200 Gy and modulation in pro-

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