Acta Physiol Plant (2014) 36:3137–3146 DOI 10.1007/s11738-014-1654-1
ORIGINAL PAPER
Reactive oxygen species generation and antioxidant defense system in hydroponically grown wheat (Triticum aestivum) upon b-pinene exposure: an early time course assessment Nadia Chowhan • Aditi Shreeya Bali • Harminder Pal Singh • Daizy R. Batish Ravinder Kumar Kohli
•
Received: 2 August 2013 / Revised: 29 July 2014 / Accepted: 22 August 2014 / Published online: 25 September 2014 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2014
Abstract We investigated the effect of b-pinene on reactive oxygen species (ROS: lipid peroxidation, membrane integrity, hydrogen peroxide and superoxide ions) generation and activity of antioxidant defense system during early hours of treatment (4, 8, 16 and 24 h) in hydroponically grown Triticum aestivum (wheat). b-Pinene reduced the root and shoot growth of the hydroponically grown wheat. However, the reduction was more pronounced in root length than in shoot length. b-Pinene enhanced ROS generation as indicated by increased levels of malondialdehyde (20–87 %), hydrogen peroxide (9–45 %) and superoxide ion (23–179 %) content, thereby suggesting lipid peroxidation and induction of oxidative stress in a time- and concentration-dependent manner. The oxidative damage was more pronounced at C10 lM bpinene and at C8 h after exposure. b-Pinene caused a severe electrolyte leakage from wheat roots indicating membrane disruption and loss of integrity. Enhanced lipid peroxidation and loss of membrane integrity were confirmed by in situ histochemical studies. b-Pinene provoked increase in the activity of lipoxygenase and upregulation in the activities of antioxidant enzymes: catalases, superoxide
Communicated by G. Bartosz. N. Chowhan A. S. Bali D. R. Batish (&) R. K. Kohli Department of Botany, Panjab University, Chandigarh 160014, India e-mail:
[email protected] H. P. Singh Department of Environment Studies, Panjab University, Chandigarh 160014, India
dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases. The enhanced activity of lipoxygenases evoked by b-pinene paralleled higher accumulation of MDA, thereby suggesting that antioxidant defense mechanism was not able to prevent b-pineneinduced lipid peroxidation. Keywords Oxygenated monoterpene Oxidative damage ROS generation Scavenging mechanism Membrane disruption
Introduction Terpenes are the largest group of phytochemicals resulting from secondary metabolism. Monoterpenes, the simplest terpenes, are usually the major components of plant essential oils and are known for various chemical interactions among plants, including allelopathy (Singh et al. 2003; Dudareva et al. 2006). Monoterpenes and essential oils can strongly inhibit seed germination and reduce plant growth (Singh et al. 2009; de Almeida et al. 2010; de Martino et al. 2010; Mutlu et al. 2010; Kaur et al. 2011; Batish et al. 2012; Vasilakoglou et al. 2013). The reasons for such growth retarding effects are not well understood, though several biochemical pathways are impaired by these natural products (Ishii-Iwamoto et al. 2012). They are known to suppress root growth by killing meristematic cells and affecting the respiratory activity, interfering with the electron flow in the cytochrome pathway, resulting in the decreased ATP production, and hence the alteration of other cellular processes that are energy demanding (Mucciarelli et al. 2001; Gniazdowska and Bogatek 2005; Bakkali et al. 2008; Ishii-Iwamoto et al. 2012). Generation of reactive oxygen species (ROS) and related oxidative
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stress has also been proposed as one of the modes of action of plant growth inhibition by monoterpenes (Zunino and Zygadlo 2004; Singh et al. 2006, 2009; Mutlu et al. 2010; Kaur et al. 2012; Hsiyung et al. 2013). Among various monoterpenes, a-pinene and its isomer b-pinene are the most abundant monoterpenes in the atmosphere surrounding the forest areas in different parts of the world, including the tropics (Stephanou 2007). Hence, an in-depth study regarding their phytotoxicity and mode of action assumes great significance. Of late, bpinene has been found to inhibit germination, reduce plant growth and induce various biochemical alterations, including impairment of protein and carbohydrate metabolism (Chowhan et al. 2011) and loss of plasma membrane integrity (Chowhan et al. 2012) in 7-day-old seedlings. Previously, it has been established that rapid burst of ROS occurs immediately after stress imposition, reaching a maximum peak within 12 h (Azevedo et al. 2009). However, no such information is available regarding the disruption of oxidative metabolism (ROS generation and ROS scavenging), during the initial hours (0–24 h) of exposure to b-pinene. It is hypothesized that during the early hours of b-pinene exposure, oxidative burst may occur leading to generation of excessive ROS, loss of membrane permeability and thus solute leakage, vis-a`-vis the alterations in the antioxidant enzymatic machinery as secondary defense strategy. We, therefore, conducted a series of experiments to provide a more thorough understanding of the timing of ROS generation under b-pinene toxicity and assessed lipid peroxidation, H2O2 and superoxide ion content, activities of superoxide dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), guaiacol peroxidases (GPX), glutathione reductases (GR) and lipooxygenases (LOX) in the roots of wheat at 4, 8, 16 and 24 h after exposure.
Materials and methods Materials Healthy seeds of Triticum aestivum L. var. PBW 502 (hereafter wheat) were purchased locally from the seed store. Before use, these were surface sterilized with sodium hypochlorite (0.1 %, w/v) for 2 min, washed under running tap water for 5 min, and then rinsed with distilled water. bPinene of technical grade (purity [ 98 %) purchased from Alfa-Aesar, Lancashire, England, was used in the experiments. All other reagents and chemicals used for biochemical analysis were of technical grade and procured from Sisco Research Laboratory Pvt. Ltd., India; Sigma Co., St. Louis, USA; Merck Ltd., India; Acros, Belgium; and Loba-Chemie Pvt. Ltd., India.
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Experimental design Wheat seeds pre-imbibed for 6 h at room temperature (25 °C) were germinated on a Whatman #1 filter paper in enamel trays (32 cm 9 23 cm 9 7 cm) lined with a moist cotton wad. Three-day-old seedlings were acclimatized in distilled water for 24 h in glass beakers (500 ml capacity). Thereafter, seedlings were exposed to different concentrations of b-pinene: 0 (control), 10, 25 50 and 100 lM for 4, 8, 16 and 24 h in a growth chamber set at day/night temperature of 20/14 (±2) °C, relative humidity of 75 ± 2 %, and a photoperiod of 16 h at a photosynthetic photon flux density (PPFD) of *240 lmol photons m-2 s-1. For each treatment, including control, five independent (beakers) replicates were maintained in a randomized block manner. After 4, 8, 16 and 24 h of treatment, wheat seedlings were harvested; their root and shoot lengths were measured. Since the effect of b-pinene toxicity was greater on roots, these were excised, washed with 10 mM CaCl2 and stored at -20 °C for assessment of oxidative damage and histochemical analysis. The concentration of b-pinene (10–100 lM = 1.36–13.6 lg ml-1) used in the present investigation is much lesser than the ones used earlier (20–800 lg ml-1) by Chowhan et al. (2011). The differences are attributed to variations in experimental conditions (hydroponics in the present study compared to Petri dish in earlier study), different growth stages (emerged seedlings rather than seeds), time of exposition, and the species specificity to bpinene. Lipid peroxidation Lipid peroxidation was determined as per the method of Heath and Packer (1968) by measuring the amount of malondialdehyde (MDA), a thiobarbituric acid reactive species (TBARS). Nearly 100-mg root was homogenized in 10 ml of 0.1 % TCA (w/v) and centrifuged at 10,0009g for 10 min. One milliliter of supernatant was mixed with 4 ml of 0.5 % thiobarbituric acid (TBA) in 20 % TCA. The mixture was heated at 95 °C for 30 min, cooled over ice, and centrifuged at 10,0009g for 10 min. The absorbance of the supernatant was read at 532 nm and corrected for non-specific turbidity by subtracting the nonspecific absorbance at 600 nm. MDA content was calculated using an extinction coefficient (e) of 155 mM-1 cm-1 and expressed as nM g-1 fw. Hydrogen peroxide (H2O2) content H2O2 was estimated as per the method described by Velikova et al. (2000). Briefly, 100-mg root tissue was extracted with 10 ml TCA (0.1 %, w/v) in an ice bath and
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centrifuged at 12,0009g for 15 min. An aliquot (0.5 ml) of the supernatant was added to 0.5 ml of PO43- buffer (pH 7.0) and 1 ml of 1 M KI. The absorbance of the mixture was recorded at 390 nm. H2O2 content was determined using e = 0.28 lM-1 cm-1 and amount expressed as nM g-1 fw. Superoxide anion (O- 2 ) content O- 2 content was determined as per the method given by Misra and Fridovich (1972). Root tissue (100 mg) was homogenized in 10 ml of 0.1 M PO43- buffer (pH 7.0) in a pre-chilled pestle mortar. The contents were centrifuged at 15,0009g for 20 min at 4 °C. To 0.2 ml of supernatant was added 1.8 ml of 1 mM adrenalin (prepared in 75 mM PO43- buffer; pH 7.4). Absorbance of the mixture was read at 480 nm immediately after addition of the enzyme extract and again after 5 min. The amount of O- 2 was calculated using e = 4020 M-1cm-1 and expressed as lM g-1fw. Root membrane integrity (REL) Membrane integrity was assessed in terms of relative electrolyte leakage (REL) from the roots in the presence of b-pinene and measured as changes in electrical conductivity (EC) of the bathing medium (Singh et al. 2007). For this, roots (100 mg) were incubated in 10 ml of distilled water at 25 °C for 2 h in test tubes and initial conductivity (E1) of the bathing medium was measured. The test tubes were further boiled for 30 min to release all the ions. These were then cooled to 25 °C and the conductivity (E2) was measured again. The REL was calculated as: REL (%) = (E1/E2) 9 100.
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ROS scavenging enzymes ROS scavenging enzymes—superoxide dismutases, SOD; catalases, CAT; ascorbate peroxidases, APX; guaiacol peroxidases, GPX; glutathione reductases, GR—were estimated in root tissue in response to b-pinene treatment. Enzyme extracts were prepared by homogenizing root tissue (150 mg) with 15 ml of 0.1 M PO43- buffer (pH 7.0) in a pre-chilled pestle and mortar. The homogenates were centrifuged at 15,0009g for 25 min at 4 °C rotor temperature in a Sigma Centrifuge. The fraction of supernatant thus obtained was used for determining the activities of various enzymes. The supernatant was stored at -20 °C before enzyme assays. The enzyme activities were measured at 25 °C on a double-beam UV–VIS spectrophotometer (Model UV 1800, Shimadzu Corporation, Japan). SOD activity was assayed in terms of the photoreduction of NBT at 560 nm (Beauchamp and Fridovich 1971). A 50 % photoreduction of NBT at 25 °C was considered as 1 unit of enzyme activity. CAT activity was determined by monitoring the disappearance of H2O2 in terms of decrease in absorbance at 240 nm as per the method of Cakmak and Marschner (1992). It was calculated by using e = 39.4 mM-1 cm-1. APX activity was determined as the rate of decrease in absorbance at 290 nm and calculated using e = 2.8 mM-1 cm-1 (Nakano and Asada 1981). GPX activity was determined in terms of guaiacol oxidized by measuring increase in absorbance at 470 nm and calculated using e = 26.6 mM-1 cm-1 (Egley et al. 1983). Activity of GR was measured by following oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm and calculated using e = 6.224 mM-1 cm-1 (Foyer and Halliwell 1976). Lipoxygenases
Histochemical detection of in situ ROS In situ ROS generation was also determined histochemically in terms of lipid peroxidation and membrane integrity. Lipid peroxidation was detected as per the method given by Pompella et al. (1987). Briefly, freshly harvested roots were stained in Schiff’s reagent for 60 min until pink color appeared. The stained roots were rinsed in 0.5 % (w/v) potassium sulfite solution (K2S2O5, prepared in 0.05 M HCl) to remove the extra stain. Root plasma membrane integrity was detected by incubating roots in 10 ml of Evans Blue solution (0.025 %, w/v, in 100 lM CaCl2, pH 5.6) for 30 min (Yamamoto et al. 2001). The stained roots were washed three times with sufficient volume of distilled water, and observed under a Trinocular Stereo Zoom Microscope (Model RSM-9, Radical Instruments, Ambala Cantt, India) fitted with a digital imaging system Nikon Cool Pix 4500 (Nikon, Japan) and photographed. In situ ROS was detected in roots on the basis of color intensity.
Lipoxygenases activity was estimated at 234 nm as per the method of Axelrod et al. (1981). The specific activities of CAT, APX, GPX, GR and LOX were expressed as enzyme unit (EU) mg-1 protein, and 1 EU is the enzyme that catalyzes 1.0 mM H2O2, ascorbate, guaiacol, NADPH or Linoleic acid min-1, respectively, at 25 °C. Data analyses All studies were performed in a randomized block design (RBD) with minimum five replicates, each consisting of a single beaker (with 20 seedlings). All the experiments and enzymatic analyses were repeated. The data were analyzed by linear regression models and the significance within curves (at different concentrations at a particular time period) and among curves (i.e. at different time periods at a particular concentration) was checked at P \ 0.05. The statistical analyses were performed using SigmaPlot 8.0 and Origin 6.0 softwares.
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Results
Root length of hydroponically grown wheat declined in a time-dependent manner in response to b-pinene over that in the control (Fig. 1). Upon 4-h exposure to b-pinene (10–100 lM), root length declined by nearly 2–6 % over the control. It declined further with increasing exposure time and 4–11 % reduction was observed after 8-h exposure. Further, after 16- and 24-h exposure to 100 lM bpinene, *27 and 33 % reduction, respectively, in root length was noticed (Fig. 1a). Likewise, shoot length of hydroponically grown wheat declined in response to bpinene. However, the reduction in shoot length was less pronounced than in root length. After 8 h of exposure,
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Y4h = 6.45 _ 0.11x ; R2 = 0.910 Y8h = 6.92 _ 0.20x ; R2 = 0.983 Y16h = 8.97 _ 0.61x ; R2 = 0.994 Y24h = 10.04 _ 0.80x ; R2 = 0.983
Root Length (cm)
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(b) 11 Y4h = 6.19 _ 0.11x ; R2 = 0.944 Y8h = 6.40 _ 0.15x ; R2 = 0.972 Y16h = 8.73 _ 0.59x ; R2 = 0.988 Y24h = 9.68 _ 0.68x ; R2 = 0.977
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β -Pinene (μM) Fig. 1 Effect of b-pinene on a root and b shoot length of hydroponically grown wheat measured at 4, 8, 16 and 24 h after exposure. Vertical bars along each data point represent the standard error of the mean. Data were analyzed by linear regression. Black lines represent regression lines and R2 represents the correlation coefficient. All regressions within curves (at different concentrations at a particular time period) and among curves (i.e. at different time periods at a particular concentration) were significant at P B 0.05
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3.0 2.5 2.0 Y4h = 0.95 + 0.30x ; R2 = 0.856 Y8h = 0.97 + 0.37x ; R2 = 0.851 Y16h = 0.94 + 0.45x ; R2 = 0.853 Y24h = 0.97 + 0.54x ; R2 = 0.853
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110 100 Y4h = 77.7 + 8.31x ; R2 = 0.998 Y8h = 79.7 + 9.12x ; R2 = 0.996 Y16h = 80.5 + 9.24x ; R2 = 0.997 Y24h = 81.1 + 9.31x ; R2 = 0.998
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Y4h = 19.79 + 3.18x ; R2 = 0.946 Y8h = 20.22 + 3.40x ; R2 = 0.952 Y16h = 20.39 + 3.51x ; R2 = 0.953 Y24h = 20.34 + 4.60x ; R2 = 0.946
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MDA content (nM g−1 fw)
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Fig. 2 Effect of b-pinene on a malondialdehyde (MDA; nM g-1 fw), -1 fw), and c hydrogen peroxide (H2O2; b superoxide ion (O- 2 ; lM g -1 nM g fw) content in the roots of hydroponically grown wheat measured at 4, 8, 16 and 24 h after exposure. Vertical bars along each data point represent the standard error of the mean. Data were analyzed by linear regression. Black lines represent regression lines and R2 represents the correlation coefficient. All regressions within curves (at different concentrations at a particular time period) and among curves (i.e. at different time periods at a particular concentration) were significant at P B 0.05
shoot length declined in the range of 3 % (at 10 lM) to 9 % (at 100 lM) over the control. After 16 and 24 h of exposure to 100 lM b-pinene, shoot length declined by *28 % over the control (Fig. 1b).
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Fig. 3 In situ histochemical localization showing b-pinene-induced a lipid peroxidation and b loss of membrane integrity in the roots of hydroponically grown Triticum aestivum roots after 4, 8, 16 and 24 h
of exposure. At each time period, roots from left to right include: 0 (control), 10, 25, 50 and 100 lM b-pinene
Lipid peroxidation
wherein exposed roots stained darker with increasing exposure period and concentration (Fig. 3a).
The amount of lipid peroxides, measured in terms of MDA content, increased significantly with the increasing concentration and period of exposure to b-pinene (Fig. 2a). At 10 lM b-pinene, MDA content enhanced over the control by almost 8–11 % after 4, 8, 16 and 24 h of exposure. The accumulation of MDA was more at higher b-pinene concentrations. At 25 lM b-pinene, MDA content enhanced in the range of 21–28 % over the control during 4–24 h of exposure (Fig. 2a). In response to 50 lM b-pinene, MDA content enhanced over the control by 35 and 48 % after 4 and 24 h of exposure. Likewise, at 100 lM b-pinene, MDA content increased by 63 % over the control after 4 h treatment and spiked further by 86 % over the control after 24-h exposure (Fig. 2a). Enhanced lipid peroxidation upon b-pinene exposure in a time- and concentration-dependent manner was confirmed by in situ detection studies with Schiff’s reagent
O- 2 content Parallel to MDA, the amount of O- 2 increased in wheat roots with increasing concentration and time of exposure to b-pinene (Fig. 2b). After 4 h of exposure to 10, 25, 50 and 100 lM b-pinene, the level of O- 2 increased by *4, *24, *47 and *110 %, respectively, over the control. O- 2 content increased further with increasing duration of exposure and after 16 h it enhanced by 7–155 % over the control, at 10–100 lM b-pinene treatment (Fig. 2b). H2O2 content b-pinene induced greater H2O2 accumulation in wheat in a dose- and time-dependent manner (Fig. 2c). After 4-h exposure, the level of H2O2 enhanced over the control by 9–42 % at 10–100 lM b-pinene. It increased further with
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period of exposure and enhanced by 1.1 (at 10 lM) to 1.5 (at 100 lM) times over the control after 8 h of exposure (Fig. 2c). Membrane integrity There was no change in EC of the medium after 4 h of exposure to b-pinene. When roots were exposed to bpinene for 8 h, EC remained unaffected up to 25 lM; however, at 50 and 100 lM concentration, EC significantly increased compared to the control depicting possible leakage of ions from roots. Leakage was more pronounced at higher concentrations of b-pinene (Fig. 4). b-Pineneinduced damage to membrane was further confirmed by staining with Evans Blue (an indicator/measure of plasma membrane integrity). At low b-pinene treatment (10 lM), roots stained lesser compared to those from highest concentration (100 lM). Further, the intensity of the stain also increased with increasing time of exposure (Fig. 3b). Antioxidant enzymes b-pinene exposure enhanced the activities of scavenging enzymes—SOD, CAT, APX, GPX, and GR—in wheat roots in a time- and concentration-dependent manner (Fig. 5a–f). After 4 h of b-pinene treatment, activity of SOD enhanced over the control by *20–157 % in response to 10–100 lM concentration (Fig. 5a). At 8 h of b-pinene exposure, SOD activity enhanced by *146 and
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234 % over the control in response to 50 and 100 lM concentration, respectively. After 24 h, *276 % increase over the control was observed in response to 100 lM bpinene (Fig. 5a). Likewise, CAT activity increased over the control by *13, 25, 36, and 49 % upon exposure to 10, 25, 50 and 100 lM b-pinene, respectively, for 4 h (Fig. 5b). After 8 h, it increased further and was *13, 26, 38 and 52 % compared to control in response to the above concentrations. After 16 and 24 h of treatment, *14–57 % increase was noticed in CAT activity in response to 10–100 lM of b-pinene (Fig. 5b). Parallel to CAT and SOD, the specific activity of APX also increased significantly, except at 8 and 16 h after exposure to 10 lM, with increasing levels of b-pinene (Fig. 5c). APX activity enhanced over the control by 1.6- to 1.9-fold in response to treatment of 100 lM b-pinene for 4–24 h. At 100 lM bpinene APX activity enhanced by *1.8-fold over the control after 16-h treatment (Fig. 5c). GPX activity also increased upon increasing the dose and duration of bpinene treatment (Fig. 5d). Exposure to 25 lM b-pinene caused *1.3-times increase in the activity of GPX compared to control at 8–24 h after exposure. At 100 lM bpinene, GPX activity increased by *1.5 to 1.6 times over the control (Fig. 5d). After 4 h of b-pinene exposure, activity of GR increased by *11–57 % over the control in response to 10–100 lM b-pinene (Fig. 5e). After 8 h, it enhanced further and was 12–64 % greater over the control. With increasing period of exposure to b-pinene, a further increase in GR activity was noticed. After 24 h of exposure to 100 lM b-pinene, it was double of that in the control (Fig. 5e). Lipoxygenase activity
Y4h = 35.3 + 0.59x ; R2 = 0.877 Y8h = 37.6 + 2.13x ; R2 = 0.894 Y16h = 37.6 + 4.82x ; R2 = 0.799 Y24h = 39.4 + 6.17x ; R2 = 0.923
Similarly, after 4 h of b-pinene treatment, activity of LOX enhanced over the control by *20–88 % in response to 10–100 lM concentration (Fig. 5f). At 8 h of exposure to 50 and 100 lM b-pinene, LOX activity enhanced by *78 and 122 %, respectively, over the control. After 24 h of exposure to 100 lM b-pinene, *127 % increase in the activity of LOX was observed (Fig. 5f).
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β -Pinene (μM) Fig. 4 b-Pinene-induced relative electrolyte leakage (% REL) in the roots of hydroponically grown wheat measured after 4, 8, 16 and 24 h of exposure. Vertical bars along each data point represent the standard error of the mean. Data were analyzed by linear regression. Black lines represent regression lines and R2 represents the correlation coefficient. All regressions within curves (at different concentrations at a particular time period) and among curves (i.e. at different time periods at a particular concentration) were significant at P B 0.05
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The present study documented that b-pinene reduced the root and shoot length of treated wheat seedlings, which is not new, and is supported by previous studies (Chowhan et al. 2011, 2012; Vasilakoglou et al. 2013). b-Pinene disturbed the cell permeability measured in terms of increased MDA content (a byproduct of lipid peroxidation) and leakage of ions in the bathing medium. Increased ion leakage suggests disruption of membrane permeability and
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= 1.39 + 0.63x, R2 = 0.977
Y8h = 1.37 + 1.00x, R2 = 0.962 Y16h = 1.52 + 1.12x, R2 = 0.960 Y24h = 1.51 + 1.45x, R2 = 0.950
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Y4h = 3.73 + 0.45x, R2 = 0.992 Y8h = 3.85 + 0.52x, R2 = 0.991 Y16h = 3.92 + 0.55x, R2 = 0.992 Y24h = 3.94 + 0.58x, R2 = 0.994
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Y4h = 0.49 + 0.07x, R2 = 0.989 Y8h = 0.52 + 0.08x, R2 = 0.986 Y16h = 0.56 + 0.09x, R2 = 0.989 Y24h = 0.58 + 0.15x, R2 = 0.920
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Y8h = 18.47 + 3.49x, R2 = 0.888 Y16h = 18.48 + 4.09x, R2 = 0.860 Y24h = 18.75 + 4.70x, R2 = 0.872
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3.5 3.0 2.5 Y4h = 1.68 + 0.37x, R2 = 0.983 Y8h = 1.65 + 0.52x, R2 = 0.977 Y16h = 1.69 + 0.53x, R2 = 0.980 Y24h = 1.74 + 0.56x, R2 = 0.994
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Fig. 5 Effect of b-pinene on the specific activity (in EU mg-1protein) of a SOD, b CAT, c APX, d GPX, e GR, and f LOX in the roots of hydroponically grown wheat measured on 4, 8, 16 and 24 h after exposure. Vertical bars along each data point represent the standard error of the mean. Data were analyzed by linear regression. Black
lines represent regression lines and R2 represents the correlation coefficient. All regressions within curves (at different concentrations at a particular time period) and among curves (i.e. at different time periods at a particular concentration) were significant at P B 0.05
loss of membrane integrity (Duke and Kenyon 1993). Membrane disruption resulting in excessive leakage of solute/ions has been suggested as one of the possible mechanisms of action of essential oils of Mentha 9 piperata (Maffei et al. 2001), Tagetes minuta (Scrivanti et al.
(2003), Artemisia scoparia (Singh et al. 2009), Rosmarinus officinalis (Stojanovic-Radic et al. 2010), and monoterpenes such as (?)-pulegone (Maffei et al. 2001), ocimene (Scrivanti et al. 2003), a-pinene (Singh et al. 2006), bmyrcene (Singh et al. 2009; Hsiyung et al. 2013), and b-
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caryophyllene (Stojanovic-Radic et al. 2010). Essential oils and their constituent monoterpenes change the fluidity of membranes, which become abnormally permeable, thereby resulting in leakage of radicals, cyt C, Ca2? and proteins as in case of oxidative stress and bio-energetic failure (Bakkali et al. 2008). The observed change in membrane permeability may also be a consequence of impairment of energy metabolism (Ishii-Iwamoto et al. 2012) or oxidative stress (Singh et al. 2006; Mutlu et al. 2010; Pergo and IshiiIwamoto 2011). The bioassays in the present study were conducted under hydroponic conditions, a well-known technique used in biological researches (Jones 1999; Torabi et al. 2012). The technique is useful for providing healthy and better root growth with an ease of harvest without any damage to root system (Jones 1999; Hershey 2008). In wheat, this technique has been widely used for various biochemical studies (Schuerger and Laible 1994; Sandı´n-Espan˜a et al. 2003; Harris and Taylor 2013). The parallel control (without bpinene) ensured a check for morphological/physiological or biochemical alterations, if any, upon hydroponic assay. In the present study, b-pinene induced generation of ROS, as indicated by the increased amounts of MDA, H2O2 and O- 2 , thereby suggesting induction of oxidative stress. ROS generation and related oxidative stress have been suggested as one of the modes of action of plant growth inhibition by allelochemicals, including essential oils (Singh et al. 2006, 2009; Mutlu et al. 2010; Batish et al. 2012; Kaur et al. 2012; Ishii-Iwamoto et al. 2012; Hsiyung et al. 2013). Pergo and Ishii-Iwamoto (2011) observed stimulation in KCN-insensitive respiration in Ipomoea triloba in response to a-pinene, thereby suggesting enhanced ROS generation. Previously, studies have reported that monoterpenes such as 1,8-cineole, geraniol, thymol, menthol and camphor (Zunino and Zygadlo 2004), a-pinene (Singh et al. 2006; Pergo and Ishii-Iwamoto 2011), and b-myrcene (Singh et al. 2009) increased MDA content, thereby suggesting lipid peroxidation. The end products of lipid peroxidation react with biomolecules, including proteins, lipids, and nucleic acid, and damage them (Apel and Hirt 2004). Accumulation of MDA, a decomposition product formed by peroxidation of polyunsaturated fatty acids in the membranes, suggests membrane damage and further generates additional free radicals (Montillet et al. 2005). Zunino and Zygadlo (2004) found that exposure to 1,8 cineole, geraniol, thymol, menthol and camphor altered the composition of membrane in maize roots. Accumulation of H2O2 further suggests oxidative damage in wheat roots upon b-pinene treatment. These observations are corroborated by earlier findings reporting greater H2O2 accumulation in response to a-pinene (Singh et al. 2006), b-myrcene (Singh et al. 2009), and essential oils of Nepeta meyeri (Mutlu et al. 2010) and Artemisia
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scoparia (Kaur et al. 2011). H2O2 acts as a signaling molecule, helps in cellular defense against stress at low concentrations, whereas hinders the activity of –SH group containing enzymes and induces cellular damage at higher levels (Stone and Yang 2006). To counter ROS-mediated cellular disintegration, various enzymatic and non-enzymatic antioxidants are produced in cellular compartment, and protect from oxidation by quenching ROS (Apel and Hirt 2004). We observed an increase in the activities of antioxidant enzymes, SOD, CAT, APX, GPX and GR, and the enzyme lipooxygenase (LOX) in a dose-dependent manner, suggesting their upregulation under b-pinene stress. These observations are paralleled by earlier study reporting greater activity of these enzymes in response to a-pinene (Singh et al. 2006; Pergo and Ishii-Iwamoto 2011). SOD activity may be upregulated to mitigate excessive generation of O- 2 ions and thus to regulate oxidative balance of the cell (Mittler et al. 2004). Because the O2 ions and the products of peroxidation of the lipid bilayer are highly reactive and immediately toxic to the cell, maximal steady-state levels of the appropriate SODs might be required to provide adequate protection. Hence, it is conceivable that high levels of oxidative stress may result in high SOD protein turnover, to maintain SOD levels sufficient for effective protection (Scandalios 1993). Increase in the activities of CAT, GPX, and APX correlated positively with the levels of H2O2, as these enzymes consume H2O2 and reduce it to water (Apel and Hirt 2004). GR is another enzyme that along with APX is involved in scavenging H2O2 from the plant cell and both are involved in ascorbate–glutathione or Asada–Halliwell–Foyer pathway (Polle 2001). GR converts oxidized glutathione (GSSG) to reduced glutathione (GSH), a compound able to scavenge ROS (Apel and Hirt 2004). Since these antioxidant enzymes belong to various cellular compartments such as mitochondria (SOD, CAT, GR), cytosol (APX, GR, SOD), plastids (SOD, GR) or peroxisomes (CAT, SOD to a lesser extent), the changes in their activities correlate to the differential sensitivity of organelles to a variety of stresses (Bailly et al. 2001). LOX, a non-heme iron containing dioxygenase, plays a major role in generating peroxidative damage in membrane lipids (Maaleku et al. 2006). The enhanced activity of LOX paralleled the higher accumulation of MDA, thereby suggesting that antioxidant defense mechanism was not able to prevent b-pinene-induced lipid peroxidation.
Conclusions The present study concludes that b-pinene provoked an increase in ROS generation, loss of membrane permeability and activity of LOX during the early hours of treatment in a
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concentration- and time-dependent manner. b-Pinene activated antioxidant defense mechanism to counter enhanced ROS generation and lipid peroxidation. However, the upregulation of antioxidant enzymes was not able to prevent b-pinene caused peroxidation of membrane lipids. Author contribution H.P. Singh and D.R. Batish designed and planned the work. N. Chowhan conducted the experiments and collected data. H.P. Singh, D.R. Batish, N. Chowhan and Aditi Shreeya Bali analyzed the data. N. Chowhan, H.P. Singh, D.R. Batish, Aditi Shreeya Bali and R.K. Kohli contributed equally to the write up of the manuscript. Acknowledgments Nadia Chowhan and Aditi Shreeya Bali are thankful to University Grants Commission (New Delhi, India) and Department of Science and Technology (New Delhi, India) for the financial support in the form of BSR fellowship and Inspire Fellowship, respectively.
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