ORIGINAL PAPER

Reactive oxygen species generation and antioxidant defensesystem 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

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract We investigated the effect of b-pinene on

reactive oxygen species (ROS: lipid peroxidation, mem-

brane 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 pro-

nounced 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 b-

pinene 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 con-

firmed by in situ histochemical studies. b-Pinene provoked

increase in the activity of lipoxygenase and upregulation in

the activities of antioxidant enzymes: catalases, superoxide

dismutases, ascorbate peroxidases, guaiacol peroxidases

and glutathione reductases. The enhanced activity of lip-

oxygenases evoked by b-pinene paralleled higher accu-

mulation of MDA, thereby suggesting that antioxidant

defense mechanism was not able to prevent b-pinene-

induced 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 interac-

tions 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 (Mu-

cciarelli 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

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: daizybatish@yahoo.com

H. P. Singh

Department of Environment Studies, Panjab University,

Chandigarh 160014, India

123

Acta Physiol Plant (2014) 36:3137–3146

DOI 10.1007/s11738-014-1654-1

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, b-

pinene 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 reduc-

tases (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. b-

Pinene of technical grade (purity [ 98 %) purchased from

Alfa-Aesar, Lancashire, England, was used in the experi-

ments. All other reagents and chemicals used for bio-

chemical 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.

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 concen-

trations 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 inde-

pendent (beakers) replicates were maintained in a ran-

domized 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 b-

pinene.

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 non-

specific absorbance at 600 nm. MDA content was calcu-

lated 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 Ve-

likova et al. (2000). Briefly, 100-mg root tissue was

extracted with 10 ml TCA (0.1 %, w/v) in an ice bath and

3138 Acta Physiol Plant (2014) 36:3137–3146

123

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 (O2-�) content

O2-� 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 O2-� 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 conduc-

tivity (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.

Histochemical detection of in situ ROS

In situ ROS generation was also determined histochemi-

cally 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.

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 temper-

ature 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 mea-

sured at 25 �C on a double-beam UV–VIS spectropho-

tometer (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 deter-

mined 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 mea-

sured by following oxidation of nicotinamide adenine dinu-

cleotide phosphate (NADPH) at 340 nm and calculated using

e = 6.224 mM-1 cm-1 (Foyer and Halliwell 1976).

Lipoxygenases

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 concentra-

tion) was checked at P \ 0.05. The statistical analyses were

performed using SigmaPlot 8.0 and Origin 6.0 softwares.

Acta Physiol Plant (2014) 36:3137–3146 3139

123

Results

Growth studies

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 expo-

sure. Further, after 16- and 24-h exposure to 100 lM b-

pinene, *27 and 33 % reduction, respectively, in root

length was noticed (Fig. 1a). Likewise, shoot length of

hydroponically grown wheat declined in response to b-

pinene. However, the reduction in shoot length was less

pronounced than in root length. After 8 h of exposure,

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).

0 10 25 50 100

Roo

t Len

gth

(cm

)

5

6

7

8

9

10

114 h8 h16 h24 h

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

β -Pinene (μM)0 10 25 50 100

Sho

ot L

engt

h (c

m)

5

6

7

8

9

10

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

(a)

(b)

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

(b)

(a)

0 10 25 50 100

O2−.

(µM

g− 1

fw)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

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

β -Pinene (μM)0 10 25 50 100

H2O

2 co

nten

t (nM

g− 1

fw)

70

80

90

100

110

120

130

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

0 10 25 50 100

MD

A c

onte

nt (n

M g

− 1 fw

)

15

20

25

30

35

40

454 h8 h16 h24 h

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

(c)

Fig. 2 Effect of b-pinene on a malondialdehyde (MDA; nM g-1 fw),

b superoxide ion (O2-�; lM g-1 fw), and c hydrogen peroxide (H2O2;

nM g-1 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 concen-

tration) were significant at P B 0.05

3140 Acta Physiol Plant (2014) 36:3137–3146

123

Lipid peroxidation

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 con-

firmed by in situ detection studies with Schiff’s reagent

wherein exposed roots stained darker with increasing

exposure period and concentration (Fig. 3a).

O2-� content

Parallel to MDA, the amount of O2-� 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 O2-� increased by *4, *24,

*47 and *110 %, respectively, over the control. O2-�

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

(a)

4h 8h 16h 24h

(b)

4h 8h 16h 24h

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

Acta Physiol Plant (2014) 36:3137–3146 3141

123

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 b-

pinene 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-Pinene-

induced 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 con-

centration (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

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 b-

pinene (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 con-

centrations. 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 signifi-

cantly, 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 b-

pinene 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 b-

pinene treatment (Fig. 5d). Exposure to 25 lM b-pinene

caused *1.3-times increase in the activity of GPX com-

pared to control at 8–24 h after exposure. At 100 lM b-

pinene, 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 con-

trol. 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

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).

Discussion

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

β -Pinene (μM)0 10 25 50 100

% R

EL

30

40

50

60

70

804 h8 h16 h24 h

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

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

3142 Acta Physiol Plant (2014) 36:3137–3146

123

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 pip-

erata (Maffei et al. 2001), Tagetes minuta (Scrivanti et al.

(2003), Artemisia scoparia (Singh et al. 2009), Rosmarinus

officinalis (Stojanovic-Radic et al. 2010), and monoter-

penes such as (?)-pulegone (Maffei et al. 2001), ocimene

(Scrivanti et al. 2003), a-pinene (Singh et al. 2006), b-

myrcene (Singh et al. 2009; Hsiyung et al. 2013), and b-

(a)

(b)

(c)

0 10 25 50 100

CA

T (E

U m

g− 1 p

rote

in)

3.0

3.5

4.0

4.5

5.0

5.5

6.0

Y4h = 3.42 + 0.41x, R2 = 0.999

Y8h = 3.43 + 0.44x, R2 = 0.999

Y16h = 3.44 + 0.47x, R2 = 0.999

Y24h = 3.45 + 0.48x, R2 = 0.999

0 10 25 50 100

SO

D (E

U m

g− 1 p

rote

in)

1

2

3

4

5

6

7

8

94 h8 h16 h24 h

Y4h = 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

β -Pinene (μM)0 10 25 50 100

AP

X (E

U m

g− 1 p

rote

in)

15

20

25

30

35

40

45

Y4h = 17.97 + 2.98x, R2 = 0.868

Y8h = 18.47 + 3.49x, R2 = 0.888

Y16h = 18.48 + 4.09x, R2 = 0.860

Y24h = 18.75 + 4.70x, R2 = 0.872

(d)

(e)

(f)

0 10 25 50 100

GPX

(EU

mg− 1

pro

tein

)

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

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

β -Pinene (μM)0 10 25 50 100

LOX

(EU

mg− 1

pro

tein

)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

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

0 10 25 50 100

GR

(EU

mg− 1

pro

tein

)

0.4

0.6

0.8

1.0

1.2

1.4

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

Fig. 5 Effect of b-pinene on the specific activity (in EU mg-1pro-

tein) 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

Acta Physiol Plant (2014) 36:3137–3146 3143

123

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 (Bak-

kali et al. 2008). The observed change in membrane per-

meability 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 Ishii-

Iwamoto 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 tech-

nique has been widely used for various biochemical studies

(Schuerger and Laible 1994; Sandın-Espana et al. 2003;

Harris and Taylor 2013). The parallel control (without b-

pinene) 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 O2-�, 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 poly-

unsaturated fatty acids in the membranes, suggests mem-

brane 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

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, vari-

ous enzymatic and non-enzymatic antioxidants are pro-

duced 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 O2-� 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 con-

verts 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 sug-

gesting 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

3144 Acta Physiol Plant (2014) 36:3137–3146

123

concentration- and time-dependent manner. b-Pinene acti-

vated antioxidant defense mechanism to counter enhanced

ROS generation and lipid peroxidation. However, the

upregulation of antioxidant enzymes was not able to pre-

vent 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 Fellow-

ship, respectively.

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