Document 2444656

Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/aeb.html
Effect of Propyl Isothiocyanate on Antioxidant Eenzymes of Garden Cress
Seedlings Under in vitro Condition
1
1
2
Farinaz Farzadnejad and 2Roya Razavizadeh
MSc student, Department of Biology, Payame Noor University, Najafabad, Isfahan, Iran
Assistant professor, Department of Biology, Payame Noor University, PO BOX 19395-3697 Tehran, Iran
ARTICLE INFO
Article history:
Received 12 November 2013
Received in revised form 19
February 2014
Accepted 26 March 2013
Available online 2 April 2014
Keyword:
vitro culture, propyl isothiocyanate,
oxidative stress
ABSTRACT
In this study effects of different levels of propyl isothiocyanate on some physiological
parameters and antioxidant enzyme activities such as ascorbate peroxidase, catalase and
guaiacol peroxidase in seedlings of Lepidiun sativum L. under in vitro was investigated.
The seeds of Lepidium sativum (Garden Cress) were sterilized and cultured in MS
medium. After 20 days, seedlings were treated with concentrations of 0.01, 0.1 and 1
mM propyl isothiocyanate (various levels of stress) under sterile condition. After 3
days, result showed chlorophyll a, chlorophyll b, total chlorophyll, carotenoids and
soluble proteins were significantly decreased, while reduced carbohydrates, proline and
antioxidant enzymes activities were increased in seedlings which have been under
oxidative stress. One of the major tissue damage following exposure to stress in plants
is caused by the oxidative stress. Thus propyl isothiocyanate appears to cause oxidative
stress and activation of the plant's defenses parameters.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Farinaz Farzadnejad and Roya Razavizadeh., Effect of Propyl Isothiocyanate on Antioxidant Eenzymes of Garden
Cress Seedlings Under in vitro Condition. Adv. Environ. Biol., 8(2), 388-395, 2014
INTRODUCTION
Inside the cell hydrolysis of glucosinolate by myrosinase enzyme (thioglucoside glucohydrolase) produces a
spectrum of products which the most important of them is isothiocyanate. Recently, the biochemical and genetic
studies performed on the Arabidospis plant confirm the existence of amino acid precursor in the glucosinolate
biosynthesis pathway. Glucosinolate exist in all of the cells (vacuoles) in different densities and in the shoots of
all members of Brassicaceae family. Glucosinolates are organic anions include D- thioglucose and
sulfonatedoxime that form an important and unique group of secondary metabolites in seeds, roots, and the
leaves of plants. When glucosinolates are adjacent myrosinase enzyme (after mechanical injury or ulcer), the
enzymes cause the hydrolysis of glucosinolate compounds in the presence of water. Hydrolysis products include
aglycone, glucose, and sulfate. The aglycone part is unstable and for the formation of isothiocyanate,
thiocyanates, nitrils, ocsasolydintions, epinitrils are rearranged based on reaction glucosinolates. Isothiocyanate
have the functional group of N=C=S. Since isothiocyanates react with amino group and sulfhydryl peptidase, it
is probable that they influence the function of peptides [34]. Most of the natural isothiocyanate in plants are
derived from chemical changes caused by glucosinolate. Natural isothiocyanate like allyl isothiocyanates are
used as flavors, aroma and fungicides [34]. These compounds have a wide range of environmental activities
including anti-oxidant, anti-bacteria, anti- fungal, anti-nematode, and anti-insect activities [34]. Isothiocyanates
are highly reactive, so they cause oxidative reactions in plant and produce active oxygen [34]. In fact, these
compounds have a double role; in high densities they have poisonous effects on cells [12]. But in lower densities
indirectly cause cell defense. Vitamin C, vitamin E and carotenoids are direct anti-oxidants and they neutralize
free radicals before they can damage the cells. Glucosinolate and their hydrolysis products are considered as
indirect anti-oxidants, because they don’t directly neutralize free radicals, they act through regulation of the
activities of xenobiotic metabolizing enzymes (phase 1 and phase 2 enzymes which launch delayed anti-oxidant
activities). Phase 1 enzymes include cytochrome P450, and the enzymes of phase 2 are glutathione Stransferase, aldehyde reductase, S-methyl transferase, N-acetyl transferase). Usually stresses disturb the cellular
electron transfer in different compartments and result in producing reactive oxygen species (ROS). One of the
important tissue injuries that are created by placing plants under stress is the increase of different kinds of
reactive oxygen and the creation of oxidative stress. Most of the metabolic processes produce reactive oxygen
species. Plant cells and their organelles like chloroplast, mitochondria and peroxisome use anti-oxidant defense
Corresponding Author: Farinaz Farzadnejad, MSc student, Department of Biology, Payame Noor University, Najafabad,
Isfahan, Iran.
E-mail: [email protected]
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Farinaz Farzadnejad and Roya Razavizadeh, 2014
Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
system to protect themselves against poisonous oxygen [7]. It seems that the ability of high plants in scavenging
radicals of poisonous oxygen is a significant factor to tolerate environmental stresses. Performed experiments
under in vitro condition show that most of the enzymes and secondary compounds protect the plants against
oxidative injuries. Study of anti-oxidant enzymes and determining the amount of their activities is one of the
determining factors in reinforcing antioxidant system and as a result decrease or increase of plant resistance to
environmental stresses [7]. Despite the existence of few reports on isothiocyanate, physiological mechanism of
plant against these compounds is not completely clear [12,14]. So, in this research the effect of propyl
isothiocyanate on physiological responses and the activities of catalase, ascorbate peroxidase and guaiacol
peroxidase in cress seedlings were studied. Isothiocyanate are among the compounds of Brassicaceae family
and have a strong smell and taste. Garden cress is also an edible plant and is called scientifically
(Lepidiumsativum L.) a one-year-old plant that its glabrous and toothless leaves are consumed by humans [28].
Plants utilize several mechanisms to respond the stresses and regarding the fact that plant mechanism is not clear
in response to isothiocyanate, this research is performed to study the effective role of isothiocyanate compounds
in defense responses through induction of antioxidant system of plant and finding the most appropriate density
of these compounds.
MATERIALS AND METHODS
The mature and sterilized seeds of cress (Lepidium sativum L.) were grown on MS medium [15]. All
cultures then were kept in the culture room with a 16/8- h light/dark photoperiod at 25 ± 2 ºC for 20 days.
Aqueous emulsion of propyl isothiocyanate at concentrations: 0, 0.01, 0.1, and 1 mM were prepared and
aseptically (cabinet laminar) using a sterile syringe was treated on seedlings in containers. Plants treated with
isothiocyanate then cultured for 3 days at the culture room. After 3 days, seedlings were frozen in liquid
nitrogen and freeze at - 80 º C and were used to measure physiological and biochemical parameters.
Measurement of chlorophyll and carotenoids:
Measurement of chlorophyll and carotenoids was determined according to [15]. 0.05 g of frozen leaf was
homogenized in 10 ml acetone 80%. This solution contains chlorophyll a, b and carotenoids. The absorbance of
each sample was read at 646.8, 663.20 and 470 nm. The amount of chlorophylls and carotenoids was expressed
as mg g-1 FW.
Measurement of anthocyanin:
Measurement of total anthocyanin was determined according to modified [33]. method using acidified
ethanol (Ethanol: HCl 99: 1 v/v). 0.05 g of frozen leaf was homogenized in 2.5 ml acidified ethanol and then
kept at 25°C for 24 h in the dark. The extract was centrifuged at 4000 g for 10 min at room temperature. The
absorbance of each supernatant was read at 550 nm. The extinction coefficient 33,000 (mol-1 cm-1) was used to
calculate the amount of total anthocyanin and it was expressed as μ mol g-1 FW.
Measurement of proline:
Proline content was estimated using ninhydrin reaction [4]. A small portion (0.5 g) of leaves or roots was
homogenized with 10 ml of 3% (w/v) sulphosalicylic acid, and passed through Whatman filter paper no. 2. Then
ninhydrin reagent (2 ml) (Sigma) and glacial acetic acid (2 ml) were added to 2 ml of the filtered extract. The
mixture was incubated at 100°C for 1 h, and the reaction was terminated by placing it on ice. The reaction
mixture was extracted with 4 ml toluene, and absorption of chromophore was measured at 520 nm, against
toluene as blank, using spectrophotometer (Shimadzu UV-160, Japan). Proline content was calculated using Lproline (Sigma) as a standard curve.
Measuring the amount of reducing carbohydrates:
Reducing carbohydrates content was measured by adapting Somogyi-Nelson’s method [30]. Approximately
0.05 g of fresh leaves and roots were extracted with 10 ml distilled water. The mixture was boiled in a boiling
water bath, cooled and filtered. Then 2 ml of the extract was mixed with 2 ml of alkaline copper tartarate and the
reaction mixture was heated for 20 min (Alkaline copper tartarate was prepared by dissolving 4 g anhydrous
sodium carbonate, 0.75 g tartaric acid and 0.45 g hydrated cupric sulphate in 80 ml of distilled water and finally
made up to 100 ml). Two ml of phosphomolibdate solution was added and the intensity of blue color was
measured at 600 nm using spectrophotometer. D-glucose was used as standard. The reducing sugar content was
expressed as mg/g FW.
Enzyme extraction and assay:
For enzyme extraction 0.1 g of shoot was homogenized using a mortar and pestle with 1 ml of 100 mM
sodium phosphate buffer (pH 7.8) containing 0.1 mM EDTA and 1% polyvinylpyrrolidone (PVP). The whole
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Farinaz Farzadnejad and Roya Razavizadeh, 2014
Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
extraction procedure was carried out on ice. The homogenates were then centrifuged for 30 min at 14000 rpm at
4°C and supernatants were used for protein and enzyme activity measurement.
Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined according to the method of Nakano and
Asada [21]. The reaction buffer for APX activity contained 50 mM sodium phosphate buffer (pH 7), 0.5 mM
ascorbic acid, 0.1 mM EDTA, 1.25 mM H2O2 and 0.05 ml enzyme extract in a final volume of 1 ml. Ascorbate
oxidation was measured at 290 nm for 1 min with extinction coefficient of 2.8 mM-1 cm-1.
Catalase (CAT, EC 1.11.1.6) activity assay was also carried out according to the method of the method of
Aebi (1984) [1]. The decrease in H2O2 was measured at 240 nm and activity was calculated as H2O2 μM
consumed per minute (extinction coefficient 39.4 mM-1 cm-1).
Guaiacol peroxidase enzyme activity was determined according to Lin and kao [16]. The reaction buffer
containing 50 mM phosphate buffer (pH =7), Guaiacol 9 mM and 19 mM hydrogen peroxide. Absorption at
wavelength of 470 nm was measured for 1 min with extinction coefficient of 6.26 mM-1 cm-1.
Determination of protein content:
The amount of total soluble protein of shoot was measured according to [5]. Absorption intensity of
extractions was determined in wave length 595 nm and the results were reported according to mg/g FW.
Statistical Analysis:
All experiments were carried out in three replications and mean values ± standard deviation were presented.
Data were subjected to ANOVA using the statistical package SPSS and the mean differences were compared by
Dunkan test at p < 0.05.
Results:
Chlorophyll a, b, total chlorophyll and carotenoids:
The amount of chlorophyll a, b and total chlorophyll and carotenoids significantly reduced under propyl
isothiocyanate stress. Concentrations of 1 and 0.1 and 0.01 mM propyl isothiocyanate decreased chlorophyll a ,
respectively 72, 55 and 51% compared with control (Figure a1). Propyl isothiocyanate at 1 mM reduced
chlorophyll b to 40% compared to controls , and the concentration of 0.1 and 0.01 respectively decreased 45 and
42 percent of chlorophyll b compared with controls (Fig. b1). The amount of total chlorophyll at concentration
of 1, 0.1 and 0.01 mM propyl isothiocyanate than control seedlings respectively 62 , 51 and 40 % decreased
(Figure c 1). Propyl isothiocyanate treatment caused significant reduction in the amount of carotenoids (Fig. d
1).
Anthocyanin content:
Anthocyanin content of leaf increased under propylisothiocyanate stress compared with control.
Propylisothiocyanate at concentrations of 1 and 0.1 mM has increased anthocyanin content of leaf 54.1 and
24.1 compared to control. Concentration of 0.01 mM showed no significant differences.
The amount of reducing carbohydrates:
Propylisothiocyanate at concentrations of 1, 0.1 and 0.01 mM caused a significant increase in sugars
respectively 4.4, 8.3 and 2.3 times compared with control in the shoot. Sugars in the roots at concentrations of 1,
0.1and 0.01 mM propylisothiocyanate increased significantly respectively 78.1, 77.1 and 46.1 percent
compared to controls.The highest amount of sugar in 1 mM and the lowest amount of sugar at 0.01 mM
propylisothiocyanate was observed.
Proline content of root and shoot:
Proline of roots increased compared to control at 1 mM propylisothiocyanate. Also, Proline of shoot
increased compared to control. Significant differences between treatments in root proline at 0.1 and 0.01 mM
was observed. Propylisothiocyanate at concentrations of 1 and 0.1 mM increased leaf proline respectively 6.1,
3.1 percent as compared to control (Figure 4).
Antioxidant enzymes activities in shoots:
The activity of all enzymes increased under propylisothiocyanate stress. Significant increase in catalase
activity at concentrations 0.1 and 0.01 mM isothiocyanate compared to controls was observed. The lowest
catalase activity at 1 mM and the highest catalase activity at 0.01 mM was obtained (Figure a 5). Ascorbat
peroxidase activity at concentration of 0.01, 0.1, and 1 mM propyl isothiocyanate, respectively, 6/5, 4/2 and 8/1
fold against the controls increased (Figure b 5). At 0.01, 0.1 and 1 mM propylisothiocyanate, activity of
guaiacoleperoxidase in leaves, respectively 2.6 , 4 and 3-fold increased compared with the control seedlings. At
a concentration of 0.01 mM, maximum enzyme activity was observed (Fig. c 5).
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Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
Fig. 1: Effect of Propyl isothiocyanate on the amount of chlorophyll a (a), chlorophyll b (b), total chlorophyll
(c) and carotenoids of Lepidium sativum (d). Values represent the mean of three replicates and
dissimilar letters are significantly different according to Duncan's test. (P ≤ 005).
Fig. 2: Effect of Propyl isothiocyanate on the amount of anthocyanin in Lepidium sativum. Values represent
the mean of three replicates and dissimilar letters are significantly different according to Duncan's test.
(P ≤ 0.05).
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Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
Fig. 3: Effect of Propyl isothiocyanate on the amount of reducing carbohydrates in Lepidium sativum. Values
represent the mean of three replicates and dissimilar letters are significantly different according to
Duncan's test. (P ≤ 0.05).
Fig. 4: Effect of propylisothiocyanate on root (a) and shoot proline (b) of Lepidium sativum. Values represent
the mean of three replicates and dissimilar letters are significantly different according to Duncan's test.
(P ≤ 0.05).
Fig. 4: Effect of propyl isothiocyanate on root (a) and shoot proline (b) of Lepidium sativum. Values represent
the mean of three replicates and dissimilar letters are significantly different according to Duncan's test.
(P ≤ 0.05)
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Farinaz Farzadnejad and Roya Razavizadeh, 2014
Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
Protein content of shoot:
The amount of protein of shoot in all treated seedlings by propylisothiocyanate than the control group
decreased. At concentrations of 1, 0.1 and 0.01 mM, reduced protein levels, respectively 40, 20 and 10%
compared to control.
Fig. 6: Effect of propylisothiocyanate on total protein content of leaf of Lepidium sativum. Values represent the
mean of three replicates and dissimilar letters are significantly different according to Duncan's test. (P ≤
0.05).
Discussion:
The findings of this research indicate that propyl isothiocyanate stress induces physiological responses and
activates antioxidant enzymes in cress seedlings. The amount of chlorophyll in plants is often estimated to
evaluate the effects of environmental tensions. These stresses may stop metabolic processes by preventing
enzyme activity. The reduction of chlorophyll in plants under stress is probably either because of controlling
chlorophyll synthesis enzymes activity or the increasing of chlorophyll pigment disintegration [24]. Propyl
isothiocyanate treatment in concentrations of 1, 0.1, 0.01 mM significantly reduced total chlorophyll of the cress
seedlings compared to control [12]. Also, it was reported that propyl isothiocyanate treatment causes
chlorophyll reduction (about 50%) in five-week- old seedlings of arabiodopsis shoot [12]. Furthermore, in the
present study propyl isothiocyanate has decreased the amount of carotenoids in leaf rather than the control
seedlings. The free radicals produced during stress cause disintegration of photosynthesis and nonphotosynthesis pigment as a result of which the pigments are decreased [24]. Carotenoids cause the protection of
photosynthesis system against extra photons and oxidative stress such as reaction to chlorophyll to prevent the
formation of active radicals of oxygen. In fact, carotenoids as a protective system against induced oxidative
stress are disintegrated and destroyed. The photochemical suppression of induced chlorophylls by carotenoids
results in the disruption of carotenoid structure and finally reduction in their amount [26]. So it is possible that
the reduction of carotenoid amount under propyl isothiocyanate treatment is caused by the role of these
pigments in protection of leaf chlorophyll against oxidative stress caused by isothiocyanats.
In this research, it is found that propyl isothiocyanate treatment has increased the amount of anthocyanin in
cress seedling leaf compared to the control seedlings. Flavonoids are polyphonic compounds and are among the
most important secondary components of plants. These components are derivatives of phenyl propanoid.
Anthocyanin as a group of soluble flavonoids is synthesized in the pathway of flavonoid biosynthesis at the end
point [17]. By creating oxidative stress in plant, antioxidant genes expression [31] and induction of phenyl
propanoid pathway especially Flavonoid biosynthesis are increased [11]. In this study, a significant difference in
amount of anthocyanin is not observed at 0.01 propyl isothiocynats and the control, but at 1 and 0.1 mM propyl
isothiocyanate, the amount of anthocyanin increased by 58 and 25 percent respectively. The same result is
reported about the increase of flavonoids during environmental stress [20]. In this research, it seems that propyl
isothiocyanate treatment causes removing of free radicals of oxygen and also plant adaptation to stress condition
by increasing anthocyanin and flavonoids. In a research, [6] it was reported that antioxidant enzyme activities
and anthocyanin content in blueberries are powerfully in contact with each other and are increased in cold
weather[30]. Also Solecka et al (1999) studied effect of low temperature stress on anthocyanin content in
cabbage. Their results showed the increase of anthocyanin compared to control seedlings. The amount of
carbohydrate and proline in the seedling root and shoot under the stress of isothiocynats showed a significant
increase compared to the control plants. To retain ion balance and osmotic regulation in vacuoles and
cytoplasm, the plants accumulate low molecular weight compounds such as proline, glycine, betaine and sugars
like glucose and fructose that are collectively called asmolite [20]. Furthermore, it seems that soluble sugars
play an important role in relation with reactive oxygen species. Sugars are also needed for performing antioxidative processes like pentose phosphate pathway [3,8] and carotenoid biosynthesis. The reduction of saved
starch and the increase of sugars are reported under stress situations [22]. Analysis of gene transcription has
verified that sugar signaling is associated with the oxidative stress control. Different stresses like salinity,
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Farinaz Farzadnejad and Roya Razavizadeh, 2014
Advances in Environmental Biology, 8(2) February 2014, Pages: 388-395
drought, low temperature and heavy metal toxicity that directly or indirectly cause the accumulation of reactive
oxygen species lead to the accumulation of soluble sugars that act as an adaptive mechanism to stress condition
[23]. In the present study, it seems that the increase of carbohydrate is because of dealing with oxygen radicals
generated under isotyocyanate stress. Proline plays an important role in stress tolerance in plant like anti-oxidant
activity of proline. Proline can remove singlet oxygen and it also can play a role in protecting proteins against
denaturation [2]. The results gained from measuring antioxidant enzyme activity under isothiosyanate stress
show that propyl isothiocynats causes an increase in the activity of catalase enzyme and more tolerance to stress.
The highest amount of catalase activity was observed at 0.01 mM of propyl isothiocynats. The balance between
producing reactive oxygen species and anti-oxidant enzyme activity determines the way that oxidative signals
occur [19]. Induction of antioxidant enzyme activity is a general adaptation of a plant against oxidative stresses
[10]. Catalase is the most important enzyme for removing oxygen peroxide and it happens by dividing it into
water and oxygen. Induction of catalase activity results in overcoming oxidative stress through hydrogen
peroxide detoxification [18]. In high concentrations of propyl isothiocynats, catalase enzyme may break by
proteases induced by oxidative stress that is reported in old pea leaves [25]. Decrease in antioxidant enzyme
activities in high levels of stress, can be caused through enzyme molecular breakage by free radicals of oxygen
[25]. Based on the results of this research, propyl isothiocynats has also increased the activity of ascorbate
peroxidase enzyme compared to the control seedlings in shoots. The highest activity of this enzyme is observed
at 0.01 mM of propyl isothiocynats. The increase of ascorbate peroxidase activity by abiotic stresses can be a
sign of the beginning of antioxidant defense. Ascorbate peroxidase enzyme has participated in ascorbateglutathione cycle and so has caused the removal of hydrogen peroxide. One study showed that allyl
isothiocynats treatment at first causes the increase and then the decrease of enzyme activity of ascorbatperoxidase in blueberries [33]. Enzyme activity of gayacule peroxidase has increased under different
concentrations of isothiocynats. Gayacule peroxidase enzyme catalyzes hydrogen peroxide- dependent oxidation
of the substrate. This enzyme also stimulates lignin biosynthesis and creates physical barrier against the stresses
and protects tissues against the free active radicals [9]. In the present research, the decrease in the activities of
these enzymes in high concentrations of propyl isothiocynats may occur through inhibition of the enzyme or
inactivation or down regulation of them so that the high intensity stress has opposite effect on the activity of the
enzyme. The other results of this research include reduction of the amount of soluble proteins in plants under
isothiocynats stress. One of the reasons of protein decrease in this level of stress might be increased production
of free radicals of oxygen. These radicals inhibit protein synthesis or lead to protein denaturation [27]. It can
also be due to reduced photosynthesis, and reduced materials required for protein synthesis under stress
conditions [13].
Conclusion:
The results of this study showed that oxidative stress induced by moderate doses of propyl isothiocynates,
induced physiological responses and antioxidant defense of cress seedlings under in vitro condition .It can be
important in plant adapt and cope against stress condition. Probably, propyl isothiocynats in cress seedlings
mediates the responses related to defense system of anti-oxidant.
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