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Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 4 ) 1 e9
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Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation Rajiv Dwivedi a, Vijay Pratap Singh b, Jitendra Kumar a, Sheo Mohan Prasad a,* a
Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad 211 002, India b Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea 497335, Chhattisgarh, India
article info
abstract
Article history:
Differential physiological and biochemical responses of two Vigna spp. i.e. Vigna mungo (L.)
Received 14 August 2014
and Vigna acontifolia (Jacq.) seedlings exposed to enhanced ultraviolet-B (ambi-
Received in revised form
entþsupplemental, 280e320 nm) radiation were studied. UV-B radiation accelerated the
2 December 2014
generation of ROS i.e. superoxide radical (O2e), hydrogen peroxide (H2O2) and hydroxyl
Accepted 10 December 2014
radical (OH) in leaves, and concomitantly damaging effects on lipid peroxidation, elec-
Available online xxx
trolyte leakage and growth in both Vigna spp. were noticed in dose dependent manner, but V. mungo exhibited greater UV-B damaging effects. UV-B stress induced positive response
Keywords:
on antioxidants: superoxide dismutase (SOD) and guaiacol peroxidase (GPX) activity, and
Vigna mungo
contents of proline, ascorbic acid, total phenolic contents (TPCs) and total flavonoid con-
Vigna acontifolia
tents (TFCs) in leaves of both spp., however, catalase (CAT) exhibited varied activity. The
UV-B radiation
study concludes that substantially higher contents of TPCs and TFCs in epidermal layer,
Reactive oxygen species
proline and ascorbic acid, and higher CAT activity before and after enhanced UV-B expo-
Lipid peroxidation
sure probably attributed greater tolerance to V. acontifolia species, thus exhibited lesser UV-
Antioxidants
B induced damaging effects on cellular components and growth than that of V. mungo. This study also suggests that V. acontifolia is comparatively resistant to UV-B and thus may be useful for practical cultivation. Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.
Introduction
Though due to the successful implementation of Montreal Protocol on substances that deplete the ozone layer, there has been a reduction in incoming solar UV-B radiation (WMO,
2010), however, terrestrial ecosystems appear to be still sen, sitive due to the variations in UV-B irradiance (Ballare Caldwell, Flint, Robinson, & Bornman, 2011). Among the living organisms, plants are vulnerable to increased UV-B radiation as they are absolutely dependent on solar radiation for their survival. Several biologically active molecules such as
* Corresponding author. Tel.: þ91 5322462048, þ91 9450609911 (mobile). E-mail addresses: [email protected] (V.P. Singh), [email protected] (S.M. Prasad). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2014.12.002 1687-8507/Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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nucleic acids, proteins and lipids absorb the high energy UV-B radiation directly and get damaged (Landry, Chapple, & Last, 1995). The findings have shown that UV-B radiation can affect growth and physiological processes in plants (Musil, Chimphango, & Dakora, 2002; Mishra, Srivastava, & Prasad, 2009). Reduction in growth of photoautotroph may occur directly due to the effects of UV-B on various cell components or indirectly through enhanced generation of reactive oxygen species (ROS). The photosynthetic and respiratory electron transport systems, photorespiratory pathway and plasma membrane have been considered as potential sources of ROS and oxidative burst (Asada, 1999). ROS play various roles in cellular system that may be positive and related to the regulation of cell growth, intercellular signaling and synthesis of biologically important compounds (Mahalingam & Fedoroff, 2003). However, at elevated concentration, ROS can be extremely harmful to organisms (Jordan, 1996). Superoxide radical (O2e) is short lived and moderately reactive ROS which is readily dismutated to relatively long-lived hydrogen peroxide (H2O2). Hydrogen peroxide may diffuse some distance from its production site and at elevated concentration inhibits photosynthesis, and thus its scavenging is vital for the proper functioning of chloroplast (Foyer & Lelandais, 1996). Superoxide and H2O2 at physiological concentration do not produce negative effects, however, their toxicity arises due to formation of metal ion dependent production of hydroxyl radicals (OH), which are capable of mutating DNA and initiating chain reaction of lipid peroxidation leading to loss of function and tissue destruction (Alscher, Erturk, & Heath, 2002). To mitigate harmful effects of ROS, the aerobic organisms have been equipped with fine regulatory mechanisms to control their levels under limit. The antioxidant defense systems successfully scavenge ROS and protect cells from oxidative damage. The enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT) and peroxidases (PODs) are efficient scavengers of O2e and H2O2. The enzyme SOD is present in all sub-cellular components susceptible to oxidative stress and determines the concentration of O2e and H2O2, and is therefore called first line of defense against ROS (Alscher et al., 2002). The scavenging of H2O2 is performed by CAT and a number of PODs. CAT decomposes H2O2 to water and molecular oxygen without consuming reductant, thus provides cells with an efficient mechanism to remove H2O2 (Scandalios, 1994). PODs are monomeric haemoproteins that catalyze the oxidation of a range of substrates by H2O2. In addition to enzymatic antioxidants, organism also contains an important array of non-enzymatic antioxidants i.e. ascorbic acid, proline, phenols, flavonoids, glutathione etc. c ik, Klejdus, Stork, (Buettner & Jurkiewiez, 1996; Kova & ovska , 2011). Malc In recent time, gradual change in environment such as enhanced UV-B radiation on the Earth's surface has become an unavoidable fact and thus, causing real threat to the existing organisms. The chlorofluorocarbons, which can deplete the ozone layer and can remain in the upper atmosphere for 40e150 years, hence, the global UV-B radiation will not recover to the levels of the pre-industrialization era by the 2050, even if all the nations implement the Montreal Protocol.
Furthermore, it is known that organisms may show differential responses to the stress factors which can be explained on the basis of their morphological, genetical, biochemical and physiological features. In the present study, an attempt has been made to understand the differential responses of growth, oxidative stress and antioxidants system in two species of Vigna i.e. Vigna mungo and Vigna acontifolia at their early stage of growth against enhanced UV-B radiation. The study is significant because (i) the crop is important in maintaining the nitrogen economy of agricultural fields and nutrient value in vegetarian diet due to high protein content, and also (ii) the early stage of seedlings that play a decisive role in the crop yield being used as vegetables and pulses appears to be most vulnerable stage of growth to enhanced UV-B radiation.
2.
Materials and methods
2.1.
Plant materials and growth conditions
The seeds of V. mungo (L.) cv. IPU- 94 -1 and V. acontifolia (Jacq.) cv. RM 570 were obtained from Indian Institute of Pulses Research (IIPR), Kanpur and Regional Research Centre, Jaipur, India, respectively. Healthy seeds were surface sterilized in sodium hypochlorite solution for 15 min and washed thoroughly with sterilized double distilled water. Thereafter, seeds were soaked for 2 h in distilled water, wrapped in moistened cotton cloth and left overnight in dark for germination. The germinated seeds were sown in plastic trays containing acid washed sterilized sand and incubated in dark at 28 ± 2 C for a day. The seedlings were grown in a growth chamber at 28 ± 2 C under 13:11h light and dark periods (550 mmol photons m2 s1, PAR) with relative humidity of 60e80%. The seedlings were watered daily with double distilled water. After 3 days of growth, equal sized seedlings were gently transferred in 0.2 strength Rorison nutrient medium (pH 7.5). The nutrient medium was aerated intermittently with sterile air to avoid the anaerobic condition around roots.
2.2.
UV-B treatment
After acclimatization in nutrient medium for two days, the seedlings were given two successive exposures of UV-B on 6th and 7th day. UV-B radiation was provided by fluorescent UV-B tube (TL e 40 W/12, Philips, Holland) with its main output at 312 nm together with white light (550 mmol photons m2 s1, PAR). The UV-B tube was covered with 0.127 mm cellulose diacetate filters (Johnston Industrial plastics, Toronto, Canada to remove radiation below 280 nm) for enhanced UV-B (eUV-B, ambient þ supplemental UV-B) radiation. Cellulose diacetate filter was changed regularly to avoid aging effects on the spectral transmission of UV-B. The UV-B irradiance at the top of the plant under the tube was measured with Power Meter (Spectra Physics, Model 407, A-2, USA). At study place, the ambient UV-B radiation was 8.6 kJ m2 d1 on the summer solstice weighted against generalized plant response action spectrum. The plants beneath cellulose diacetate film received different levels of biologically effective UV-B radiation (UV-BBE) i.e. ambientþ1.2 kJ m2, ambientþ2.4 kJ m2, ambientþ3.6 kJ m2, ambientþ4.8 kJ m2 and
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 4 ) 1 e9
ambientþ7.2 kJ m2 simulating 4, 5, 6, 7 and 8%, respectively reduction in stratospheric ozone at Allahabad (20 470 N) during clear sky condition on the summer solstice. The various levels of enhanced UV-B radiation were achieved by managing distance between plant canopy and surface of UV-B tube. On 8th day seedlings of each set was harvested and various parameters related to growth, oxidative stress and antioxidant system were analyzed. The seedlings which were not exposed to UV-B regarded as “control”.
2.3.
Measurement of growth and relative water content
After 24 h of last UV-B exposure (on 8th day), seedlings of each set were harvested and plant fresh and dry mass was determined by single pan electronic balance (Contech- CA 223, India). For dry mass measurement, the seedlings were wrapped in butter paper and dried in an oven at 80 C for 24 h. The leaf area of treated and untreated seedlings was determined by leaf area meter (Model 211, Systronics, India). Relative water content (RWC) of leaf was determined as (FW DW)/ (TW DW) 100, where FW is the fresh weight, DW is the dry weight and TW is the turgid weight of the leaf after equilibration in distilled water for 24 h.
2.4.
Estimation of reactive oxygen species
Superoxide radical O2e was measured by the method of Elstner and Heupel (1976). This assay is based on formation of nitrite (NO 2 ) formation from hydroxylamine in the presence of O2e. The absorbance of the colored aqueous phase was recorded at 530 nm. A standard curve prepared with NaNO 2 was used to calculate the production rate of O2e. Hydrogen peroxide (H2O2) content in the treated and untreated samples was estimated by following ferrithiocyanate method as described by Sagisaka (1976). The absorbance of the solution was recorded at 480 nm, and amount of H2O2 in each sample was calculated from standard curve. Hydroxyl radical (OH) was quantified by the method of Babbs, Ann Pham, and Coolbaugh (1989), using dimethyl sulfoxide (DMSO) as molecular probe which is oxidized by hydroxyl radical into methane sulfinic acid (MSA), a stable compound. The absorbance of samples was recorded at 425 nm and an extinction coefficient 2088 M1 cm1 was used to calculate OH radicals and were expressed as MSA content.
2.5.
Estimation of oxidative damage indices
Lipid peroxidation was estimated as malondialdehyde (MDA) content adopting the method of Hodges, De Long, Forney, and Prange (1999). Malondialdehyde content was calculated using the equation: MDA (nmol ml1) ¼ (AB/157,000) 106 where A ¼ [(A532 þ TBA) (A600 þ TBA) (A532 TBA A600 TBA)] and B ¼ [(A440 þ TBA A600 þ TBA)0.0571]. Intactness of plasma membrane in leaves was measured as the leakage percentage of electrolytes as described by Gong, Li, and Chen (1998). The leakage percentage of electrolytes was calculated by using the formula (EC1/EC2) 100.
2.6.
3
Assay of enzymatic antioxidants
Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by measuring the inhibition of the reduction of p-nitroblue tetrazolium chloride (NBT) by the method of Giannopolitis and Ries (1977). The initial rate of reaction as measured by the difference in absorbance at 560 nm, in the presence and absence of extract was proportional to the amount of enzyme. One unit of SOD was defined as the amount of enzyme that inhibited NBT reduction by 50% under the specified conditions. Guaiacol peroxidase (GPX, EC 1.11.1.7) activity in leaf homogenates of treated and untreated seedlings was determined according to the method of Zhang (1992). One unit of the GPX activity is the amount of enzyme oxidizing 1 nmol guaiacol min1 at 28 C. Catalase (CAT, EC 1.11.1.6) activity was determined by following the method of Aebi (1984). One unit enzyme activity is represented as 1 mmol H2O2 decomposed min1. Protein content in each sample was estimated according to Bradford (1976).
2.7.
Estimation of non-enzymatic antioxidants
Total ascorbate was determined by the method of Gossett, Millhollon, and Cran (1994). This assay is based on reduction of Fe3þ into Fe2þ with ascorbic acid in acid solution followed by the formation of red chelate between Fe2þ and 2,2-bipyridyl. Ascorbate content was calculated by using standard curve prepared with L-ascorbic acid. Proline content in leaf homogenate of UV-B treated and untreated seedlings was estimated according to the method of Bates, Waldren, and Teare (1973). The proline content in each sample was calculated from the standard curve. Total phenolic contents (TPCs) were estimated following the method of Waterhouse (2001). The amount of TPCs was expressed as mg gallic acid equivalents g1 dry weight. Total flavonoid contents (TFCs) in treated and untreated seedlings were determined spectrophotometrically using AlCl3 method using quercetin as standard (Zhishen, Mengcheng, & Jianming, 1999). After incubating reaction mixtures at room temperature, optical density was measured at 512 nm. TFCs were expressed as mg quercetin equivalents g1 dry weight.
2.8.
Statistical analysis
Results were statistically analyzed by analysis of variance (ANOVA). Duncan's multiple range tests was applied for mean separation for significant differences among treatments using SPSS software (Version 10, SPSS Inc., Chicago). Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6).
3.
Results
3.1.
Growth
UV-B radiation induced impact on growth of V. mungo and V. acontifolia seedlings was analyzed and results are presented in
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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Table 1 e Effect of enhanced UV-B radiation on plant fresh mass (PFM, mg plant¡1), plant dry mass (PDM, mg plant¡1) and relative water content (RWC) of Vigna mungo and Vigna acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent decrease (¡) over control value. UV-B exposure
Growth parameters Vigna mungo PFM
Control Ambþ1.2 kJ Ambþ2.4 kJ Ambþ3.6 kJ Ambþ4.8 kJ Ambþ7.2 kJ
2
m m2 m2 m2 m2
324.0 310.0 298.0 281.6 267.3 251.4
± 5.37a ± 5.32 (4)b ± 2.70 (8)c ± 2.43 (13)d ± 3.84 (18)e ± 3.25 (22)f
Vigna acontifolia
PDM 27.2 26.1 24.8 23.1 22.1 20.8
RWC (%)
± 0.33a ± 0.37 (4)a ± 0.54 (9)b ± 0.25 (15)c ± 0.53 (19)cd ± 0.17 (24)ef
91.6a 91.3a 90.6a 90.2a 89.9a 89.8a
Tables 1 and 2. UV-B declined (P < 0.05) growth parameters: biomass accumulation in leaves and in whole plant and also on leaf area in dose dependent manner in both the species. Leaf area showed maximum reduction (P < 0.05) compared to other growth parameters which exhibited 35% decline in V. mungo and only 19% in V. acontifolia following Ambþ7.2 kJ m2 UV-B exposure. Similar UV-B dose decreased leaf dry mass by 29% and 19%, and plant dry mass by 24% and 20% in V. mungo and V. acontifolia, respectively. In contrast to growth parameters, RWC was not affected significantly (P < 0.05) by UV-B in both the species (Table 1).
3.2.
3.3.
PFM 206.6 201.2 193.7 186.7 179.4 172.0
± 4.0g ± 5.31 ± 4.44 ± 2.94 ± 2.64 ± 4.82
PDM 21.5 20.5 19.7 19.2 18.4 17.3
gh
(3) (6)hi (10)ij (13)jk (17)k
RWC (%)
± 0.32de ± 0.53 (5)ef ± 0.38 (8)fg ± 0.44 (11)gh ± 0.33 (14)hi ± 0.29 (20)i
89.8a 89.6a 89.5a 89.1a 88.8a 88.6a
Lipid peroxidation and membrane damage
The data presented in Fig. 2a reveal that UV-B radiation caused substantial damage to cellular membrane in both species as malondialdehyde (MDA) content, a peroxidation product of lipid raised progressively following the exposure of leaves with increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) of enhanced UV-B radiation. The accumulation of MDA content was significantly (P < 0.05) higher in V. mungo, and after Ambþ7.2 kJ m2 of UV-B exposure MDA content was enhanced by 119% and 81% in V. mungo and V. acontifolia, respectively over their respective control. Further, the oxidative stress induced damaging effect was also analyzed by determining the electrolyte leakage in the leaves of both the species. The results presented in Fig. 2b depicts that UV-B exposure with increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) caused progressive increase in electrolyte leakage in leaf tissues; however, the damaging effect was stronger (P < 0.05) in V. mungo than V. acontifolia.
Reactive oxygen species
The results pertaining to superoxide radical (O2e), hydrogen peroxide (H2O2) and hydroxyl radical (OH) contents in the leaves of UV-B exposed seedlings are depicted in Fig. 1aec. Compared to the V. acontifolia, the control seedlings of V. mungo exhibited significantly (P < 0.05) higher contents of O2e, H2O2 and OH showing 180%, 12% and 21%, respectively higher amounts than the respective values in V. acontifolia. UV-B dose (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) dependent rise in ROS contents was noticed in both species but the rate of generation/accumulation of O2e, H2O2 and OH was substantially higher in V. mungo.
3.4.
Enzymatic antioxidants
The results pertaining to superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) are shown in Table 3.
Table 2 e Effect of enhanced UV-B radiation on leaf area (LA, mm2 plant¡1), leaf fresh mass (LFM, mg plant¡1) and leaf dry mass (LDM, mg plant¡1) of Vigna mungo and Vigna acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent decrease (¡) over control value. UV-B exposure
Growth parameters Vigna mungo LA
Control Ambþ1.2 kJ Ambþ2.4 kJ Ambþ3.6 kJ Ambþ4.8 kJ Ambþ7.2 kJ
m2 m2 m2 m2 m2
348.1 329.4 317.8 274.0 254.4 227.1
± 4.46a ± 4.73 (5)b ± 4.91 (9)b ± 4.44 (21)e ± 3.57 (27)g ± 3.60 (35)h
LFM 56.4 ± 54.6 ± 52.5 ± 47.9 ± 41.3 ± 37.2 ±
0.43a 0.36 (3)b 0.32 (7)c 0.17 (15)d 0.45 (27)g 0.32 (34)i
Vigna acontifolia LDM 7.8 ± 0.19a 7.3 ± 0.18 (6)b 6.7 ± 0.20 (14)c 6.4 ± 0.19 (18)cd 6.0 ± 0.13 (23)def 5.5 ± 0.17 (29)fg
LA 319.7 ± 304.2 ± 295.6 ± 290.4 ± 269.8 ± 257.4 ±
3.16b 5.21 (5)c 4.83 (8)cd 4.84 (9)d 3.89 (16)ef 4.29 (19)fg
LFM 44.2 42.7 41.5 40.8 38.4 36.6
± 0.41e ± 0.51 (3)f ± 0.43 (6)g ± 0.23 (8)g ± 0.23 (13)h ± 0.26 (17)i
LDM 6.7 ± 6.5 ± 6.3 ± 6.1 ± 5.8 ± 5.4 ±
0.19c 0.15 (3)cd 0.18 (6)cde 0.15 (9)de 0.17 (13)efg 0.15 (19)g
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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Ambþ7.2 kJ m2), however, it was maximum in leaves of V. mungo and V. acontifolia after Ambþ2.4 kJ m2 and Ambþ4.8 kJ m2 of UV-B exposure, respectively. CAT activity in V. mungo initially (up to Ambþ2.4 kJ m2 of UV-B exposure) showed increasing trend and thereafter, it declined progressively exhibiting about 14% less activity over the value of control when leaves were irradiated with Ambþ7.2 kJ m2. In V. acontifolia CAT activity though exhibited a declining trend following the increasing UV-B (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) exposure but the activity was still higher (P < 0.05) in comparison to the V. mungo. UV-B exposure caused an accelerating effect on GPX activity in both the species and increased progressively in V. mungo while it was maximum in Ambþ4.8 kJ m2 UV-B treated leaves of V. acontifolia.
3.5.
Non-enzymatic antioxidants
The results pertaining to ascorbic acid, proline, total phenolic contents (TPCs) and total flavonoid contents (TFCs) in leaves of both the species are depicted in Fig. 3aed, respectively. The untreated leaves of V. acontifolia exhibited comparatively higher contents of non-enzymatic antioxidants showing 41%, 43%, 44% and 61% more ascorbic acid, proline, TPCs and TFCs than V. mungo. The increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2 min) of UV-B caused progressive rise in ascorbic acid and proline contents however, a steep decline (P < 0.05) in V. mungo was recorded following Ambþ7.2 kJ m2 of UV-B exposure. There was significant enhancement in TPCs and TFCs in V. mungo with the rising doses of UV-B. In V. acontifolia TFCs showed initially increasing trend (up to Ambþ2.4 kJ m2 of UV-B exposure) which was substantially high. Further, increase in UV-B dose (Ambþ3.6 kJ m2 to Ambþ7.2 kJ m2) caused a declining trend; however, the content was significantly (P < 0.05) higher compared to the values obtained in V. mungo under similar condition.
4.
Fig. 1 e Superoxide radical (a), hydrogen peroxide (b) and hydroxyl radical (c) contents in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test.
GPX activity was significantly higher in the leaves of control V. mungo leaves while CAT activity was appreciably more in V. acontifolia. SOD activity in both species exhibited an enhanced rate following UV-B exposure (Ambþ1.2 kJ m2 to
Discussion
UV-B dose dependent reduction in growth parameter (biomass accumulation) in V. mungo and V. acontifolia seedlings was recorded (Tables 1 and 2). Besides this, visual symptoms such as dwarfing of plants, distortion of shoots, and reduction in leaf dimension in the seedlings of both the species were also observed (data not shown). Similar to our results enhanced level of UV-B radiation significantly declined the growth in Vigna unguiculata (Musil et al., 2002). Such reduction in growth could be associated with UV-B induced inhibition in photosynthetic rate and destruction of growth promoting hormone: indole acetic acid (IAA) (Kulandaivelu, Maragatham, & Nedunchezhian, 1989). Further, it can also be linked with excessive generation/accumulation of free radicals (Fig. 1aec) that cause damaging effect on structural and functional components of cells. UV-B induced strong reduction in growth of Vigna species at higher dose particularly in V. mungo might have occurred due to irreparable damage to biomolecules associated with photosynthetic apparatus particularly to the membrane involved in energy transducing system and also to the C3 cycle enzymes as
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
2.33h 2.82 (þ8)h 2.67 (þ13)h 4.64 (þ50)f 3.39 (þ108)d 4.01 (þ33)g 85.6 ± 92.6 ± 96.3 ± 128.3 ± 178.1 ± 114.0 ± 28.1 ± 0.54a 25.8 ± 0.57 (8)b 23.6 ± 0.58 (16)c 21.8 ± 0.58 (22)d 19.5 ± 0.32 (31)e 17.7 ± 0.60 (37)f ± 0.16f ± 0.15 (þ5)f ± 0.15 (þ13)ef ± 0.13 (þ25)e ± 0.19 (þ28)e ± 0.15 (þ5)f 4.0 4.2 4.5 5.0 5.1 4.2 3.0g 4.07 (þ30)e 6.07 (þ90)c 6.05 (þ125)b 4.17 (þ134)ab 3.29 (þ140)a 114.8 ± 149.1 ± 218.2 ± 257.8 ± 269.2 ± 275.0 ± ± 0.65gh ± 0.50 (þ8)fg ± 0.33 (þ14)f ± 0.39 (þ8)fg ± 0.41 (8)hi ± 0.61 (14)i 15.3 16.5 17.5 16.5 14.1 13.1 0.44d 0.39 (þ28)c 0.19 (þ62)a 0.17 (þ43)b 0.31 (þ40)b 0.34 (þ24)c 10.5 ± 13.4 ± 17.0 ± 15.0 ± 14.7 ± 13.0 ± kJ m2 kJ m2 kJ m2 kJ m2 kJ m2 Control Ambþ1.2 Ambþ2.4 Ambþ3.6 Ambþ4.8 Ambþ7.2
CAT (units mg1 protein min1)
V. acontifolia
SOD (units mg1 protein) CAT (units mg1 protein min1)
V. mungo
GPX (units mg1 protein min1)
Enzyme activities
SOD (units mg1 protein)
observed in earlier findings (Bischof, Hanelt, & Wiencke, 2000; Mishra, Srivastava, Prasad, & Abraham, 2008). UV-B exposure may stimulate the generation of ROS in plants by disrupting electron transport system (ETS) and diverting the normal path of electron in the photosynthetic and respiratory electron transport systems (Halliwell & Gutteridge, 1999). Disruption of ETS in various cellular compartments resulted in enhanced generation/accumulation of ROS thus, O2e, H2O2 and OH, the potent inhibitors of a number of key metabolic processes, increased substantially in UV-B exposed Vigna species seedlings (Fig. 1aec). Compared to the V. acontifolia, the contents of O2e, H2O2 and OH were found to be appreciably higher in V. mungo control leaves and the amount of ROS increased continuously with rising doses of UV-B. The greater accumulation of ROS particularly H2O2 in V. mungo leaves could be correlated with UV-B induced substantial damaging effects on photosynthetic electron transport and CO2 fixation as reported in V. unguiculata (Mishra
UV-B exposure
Fig. 2 e Lipid peroxidation as MDA content (a) and electrolyte leakage (b) in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test.
GPX (units mg1 protein min1)
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Table 3 e Effect of enhanced UV-B radiation on antioxidant enzyme activities in leaves of V. mungo and V. acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent increase (þ) or decrease (¡) over control value.
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Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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et al., 2008). Thus, this condition probably led to the spilling of electron to O2 resulting in the enhanced generation of ROS. ROS produce damaging effects on functional biomolecules and if there were excess ROS formed within the cells, one would expect impairment in several metabolic processes that eventually lead to cell death. H2O2 at elevated level (mM concentration) is known to inactivate enzymes by oxidizing their thiol groups as reported in case of Calvin cycle enzymes (Devine, Duke, & Fedtke, 1993, ). However, the real threat of O2e and H2O2 is their potential to act as precursors for OH as we have observed increased amount of OH in UV-B dose dependent manner (Fig. 1c). The OH can readily oxidize amino acid residues of proteins, fatty acids of phospholipids and deoxyribose and bases in DNA (Halliwell & Gutteridge, 1999). In the present study, even untreated seedlings of two Vigna species showed significant (P < 0.05) differences in MDA content (Fig. 2a) which could be linked with faster rate of physiological activity in V. mungo (Tables 1 and 2) leading to more generation/accumulation of O2e, H2O2 and OH (Fig. 1aec). Further, UV-B exposure increased the MDA content in both the species of Vigna in UV-B dose dependent manner
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and it was significantly (P < 0.05) higher in UV-B exposed V. mungo leaves compared to V. acontifolia (Fig. 2a). The membrane stability of leaf discs of Vigna species seedlings also showed similar trend as observed in MDA content (Fig 2b). Lower lipid peroxidation and lower electrolyte leakage in V. acontifolia seedlings may be associated with relative tolerance of this species to UV-B, while the reverse was noticed for V. mungo seedlings. To mitigate the negative impact of oxidative stress, plants have efficient antioxidative defense system which provides adequate protection against ROS and free radicals. However, under severe stress condition the antioxidant capacity of plants may not bring the level of ROS under limit thus, oxidative injury takes place. The antioxidant enzymes induced by UV-B are likely to be species specific. Activities of SOD, CAT, APX and GPX were reported to be enhanced by UV-B radiation (Kondo & Kawashima, 2000; Mishra et al., 2009) while decreased CAT activity was reported in barley seedlings (Zancan, Suglia, La Rocca, & Ghisi, 2008). In the present study, the response of antioxidant enzymes: SOD, CAT and GPX varied with UV-B exposure and species tested (Table 3).
Fig. 3 e Ascorbic acid (a), proline (b), total phenolic contents (c) and total flavonoid contents (d) in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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Though the leaves of V. mungo seedlings exhibited higher rate of SOD and GPX activities which showed increasing trends with UV-B dose (except Ambþ7.2 kJ m2), however, there was considerable accumulation of ROS leading to enhanced damaging effects (Fig. 2a and b). The greater accumulation of the ROS (Fig. 1aec) in V. mungo probably resulted due to the imbalance between generation/accumulation of ROS and antioxidant systems. In comparison to the V. acontifolia, V. mungo before and after UV-B exposure exhibited lower CAT activity that might be partially responsible for higher accumulation of ROS (H2O2 and OH). The substantial decrease in CAT activity at high UV-B doses might have occurred either due to down regulation of the genes coding for this enzyme or ROS mediated damaging effect. In the present study, under UV-B stress, there was substantial increase in GPX activity in V. mungo seedlings (Table 3). The accelerated GPX activity can also be correlated with reduced growth and biomass accumulation as peroxidase may initiate the catabolism of growth promoting hormone like indole acetic acid (IAA) (Kulandaivelu et al., 1989), which is supported by the increased UV-B sensitivity of tobacco transgenic plants with decreased auxin levels (Lagrimini, 1999). Apart from enzymatic antioxidants, plant cells also contain an important array of non-enzymatic antioxidants such as ascorbic acid, proline, phenolic compounds, flavonoids, glutathione etc. for mitigating the toxic effects of ROS. The low molecular weight antioxidants are even helpful in scavenging those ROS (singlet oxygen; 1O2 and OH) for which no enzymatic scavenging system has been evolved. Ascorbic acid is one of the most powerful antioxidants that scavenges the H2O2 and other ROS profoundly. Protective effect of ascorbic acid is possibly more related to its participation in direct scavenging of 1O2 and OH, and also the removal of H2O2 through Asada-Halliwell pathway thus, reducing ROS induced damage to essential proteins and/or nucleic acids (Buettner & Jurkiewiez, 1996). Significantly (P < 0.05) higher ascorbic acid content in V. acontifolia leaves before (in control plants) and after UV-B exposure showed its greater capability to scavenge ROS (Fig. 3a). There may be two possibilities regarding increased ascorbic acid content; either its synthesis has been amplified or its regeneration through Asada-Halliwell pathway. Proline content has also been shown to be increased in plants exposed to various stresses including UV-B (Mahdavian, Ghorbanli, & Kalantari, 2008) and similar trend was also recorded in the present study (Fig. 3b). Further, significantly (P < 0.05) higher proline content in V. acontifolia justified its protective role against UV-B stress as noticed in earlier finding (Mahdavian et al., 2008). The phenols and flavonoids (UV-B absorbing pigments) provide the first line of defense against UV-B as they are primarily located in leaf epidermal layers, preventing its penetration to the leaf interior. The importance of flavonoids in leaf epidermal layers was confirmed by applying a fiber-optic c ik microprobe (Bornman & Vogelmann, 1991). Further, Kova et al. (2011) have reported importance of TPCs and TFCs in UV protection in a lichen- Xanthoria parietina. Compared to the V. mungo, significantly (P < 0.05) higher TPCs and TFCs in V. acontifolia leaves before and after UV-B exposure probably prevented the penetration of UV-B radiation to the leaf
interior, thus suggesting better adaptability towards UV-B stress (Fig. 3c and d).
5.
Conclusion
The study concludes that enhanced UV-B exposure caused significant reduction in growth in both the Vigna species; however, the damaging effects were more prominent in V. mungo. In spite of high SOD and GPX activities, the greater accumulation of ROS and associated membrane damage confirm the higher sensitivity of V. mungo seedlings to enhanced UV-B irradiation. Appreciably higher contents of ascorbic acid, proline, TPCs and TFCs, and comparatively more activity of CAT in V. acontifolia leaves before and after UV-B exposure render it as a more resistant species towards enhanced UV-B while the reverse is apparently true for V. mungo.
Acknowledgments Rajiv Dwivedi and Vijay Pratap Singh are thankful to CSIR and UGC, respectively for providing financial support in the form of JRF and SRF to carry out this work. The financial assistance provide to “Jitendra Kumar” as JRF under the scheme RGNF2012-13-SC-UTT-33185 funded by UGC New Delhi, India.
references
Aebi, I. I. (1984). Catalase in vitro. Methods Enzymology, 105, 121e126. Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutase (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 53, 1331e1341. Asada, K. (1999). The water - water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology Plant Molecular Biology, 50, 601e639. Babbs, C. F., Ann Pham, J., & Coolbaugh, R. C. (1989). Lethal hydroxyl radical production in paraquat-treated plants. Plant Physiology, 90, 1267e1270. , C. L., Caldwell, M. M., Flint, S. D., Robinson, S. A., & Ballare Bornman, J. F. (2011). Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change. Photochemistry and Photobiology Science, 10, 226e241. Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid determination of free proline in water-stress studies. Plant and Soil, 39, 205e207. Bischof, K., Hanelt, D., & Wiencke, C. (2000). Effect of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae. Planta, 211, 555e562. Bornman, J. F., & Vogelmann, T. C. (1991). Effect of UV-B radiation on leaf optical properties measured with fiber optics. Journal of Experimental Botany, 42, 547e554. Bradford, M. (1976). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Buettner, G. R., & Jurkiewiez, B. A. (1996). Chemistry and biochemistry of ascorbic acid. In E. Cadenas, & L. Packer (Eds.),
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 4 ) 1 e9
Handbook of antioxidants (pp. 91e115). New York: Marcel Dekker. Devine, M., Duke, S. O., & Fedtke, C. (1993). Physiology of herbicidal action. Englewood Cliffs: PTR Prentice-Hall. Elstner, E. F., & Heupel, A. (1976). Inhibition of nitrite formation from hydroxyl ammonium chloride a simple assay per superoxide dismutase. Analytical Biochemistry, 70, 616e620. Foyer, C. H., & Lelandais, M. A. (1996). A comparison of the relative rates of transport of ascorbate and glucose across the thylakoid, chloroplast and plasma membranes of pea leaves mesophyll cells. Journal of Plant Physiology, 148, 391e398. Giannopolitis, C. N., & Ries, S. K. (1977). Superoxide dismutase. I. Occurrence in higher plants. Plant Physiology, 59, 309e314. Gong, M., Li, Y. J., & Chen, S. Z. (1998). Abscisic acid induced thermotolerance in maize seedlings is mediated by calcium and associated with antioxidant system. Journal of Plant Physiology, 153, 488e496. Gossett, D. R., Millhollon, E. P., & Cran, L. M. (1994). Antioxidant response to NaCl stress in salt sensitive cultivars of cotton. Crop Science, 34, 706e714. Halliwell, B., & Gutteridge, J. M. C. (1999). Free radicals in biology and medicine (3rd ed.). Oxford: Oxford University Press. Hodges, D. M., De Long, J. M., Forney, C. F., & Prange, R. K. (1999). Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207, 604e611. Jordan, B. R. (1996). The effects of ultraviolet-B radiation on plants: a molecular perspective. Advance in Botanical Research, 22, 97e162. Kondo, N., & Kawashima, M. (2000). Enhancement of the tolerance to oxidative stress in cucumber (Cucumis sativus L.) seedlings by UV-B irradiation: possible involvement of phenolic compounds and antioxidative enzymes. Journal of Plant Research, 113, 311e317. c ik, J., Klejdus, B., Stork, ovska , S. (2011). Sensitivity Kova F., & Malc of Xanthoria parietina to UV-A: role of metabolic modulators. Journal of Photochemistry and Photobiology B: Biology, 103, 243e250. Kulandaivelu, G., Maragatham, S., & Nedunchezhian, N. (1989). On the possible control of ultraviolet eB induced response in growth and photosynthetic activities in higher plants. Physiologia Plantarum, 76, 398e404. Lagrimini, L. M. (1999). The role of peroxidase in auxin metabolism. In L. M. Lagrimini (Ed.), Abstracts peroxidase 99. International symposium for plant peroxidases (p. 27). Columbus: Ohio State University.
9
Landry, L. C., Chapple, C. C., & Last, R. L. (1995). Arabidopsis mutants lacking phenolics sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiology, 109, 1159e1166. Mahalingam, R., & Fedoroff, N. (2003). Stress response, cell death and signaling: the many faces of active oxygen species. Physiologia Plantarum, 119, 56e68. Mahdavian, K., Ghorbanli, M., & Kalantari, K. M. (2008). The effect of UV-B radiation on the content of chlorophyll, flavonoids, anthocyanin and proline in Capsicum annuum L. Turkish Journal of Botany, 32, 25e33. Mishra, V., Srivastava, G., & Prasad, S. M. (2009). Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation. Scientia Horticulturae, 120, 373e378. Mishra, V., Srivastava, G., Prasad, S. M., & Abraham, G. (2008). Growth, photosynthetic pigments and photosynthetic activity during seedling stage of cowpea (Vigna unguiculata) in response to UV-B and dimethoate. Pesticide Biochemistry and Physiology, 92, 30e37. Musil, C. F., Chimphango, S. B. M., & Dakora, F. D. (2002). Effects of elevated ultraviolet-B radiation on native and cultivated plants of southern Africa. Annals of Botany, 90, 127e137. Sagisaka, S. (1976). The occurrence of peroxide in a perennial plant Populus gelrica. Plant Physiology, 57, 308e309. Scandalios, J. G. (1994). Regulation and properties of plant catalases. In C. H. Foye, & P. M. Mullineaux (Eds.), Causes of photooxidative stress and amelioration of defense system in plants (pp. 275e315). Boca Raton: CRC Press. W.M.O. (2010). Scientific assessment of ozone depletion: 2010. Global Ozone Research and Monitoring ProjecteReport No. 52, 516 pp. Geneva, Switzerland, 2011. Waterhouse, A. L. (2001). Determination of total phenolics, handbook of food analytical chemistry, unit I 1.1: Polyphenolics (pp. 464e465). New York: Wiley. Zancan, S., Suglia, I., La Rocca, N., & Ghisi, R. (2008). Effect of UV-B radiation on antioxidant parameters of iron-deficient barley plants. Environmental and Experimental Botany, 63, 71e79. Zhang, X. Z. (1992). The measurement and mechanism of lipid peroxidation and SOD, POD and CAT activities in biological system. In X. Z. Zhang (Ed.), Research methodology of crop physiology (pp. 208e211). Beijing: Agriculture Press. Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64, 555e559.
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Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation Rajiv Dwivedi a, Vijay Pratap Singh b, Jitendra Kumar a, Sheo Mohan Prasad a,* a
Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad 211 002, India b Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea 497335, Chhattisgarh, India
article info
abstract
Article history:
Differential physiological and biochemical responses of two Vigna spp. i.e. Vigna mungo (L.)
Received 14 August 2014
and Vigna acontifolia (Jacq.) seedlings exposed to enhanced ultraviolet-B (ambi-
Received in revised form
entþsupplemental, 280e320 nm) radiation were studied. UV-B radiation accelerated the
2 December 2014
generation of ROS i.e. superoxide radical (O2e), hydrogen peroxide (H2O2) and hydroxyl
Accepted 10 December 2014
radical (OH) in leaves, and concomitantly damaging effects on lipid peroxidation, elec-
Available online xxx
trolyte leakage and growth in both Vigna spp. were noticed in dose dependent manner, but V. mungo exhibited greater UV-B damaging effects. UV-B stress induced positive response
Keywords:
on antioxidants: superoxide dismutase (SOD) and guaiacol peroxidase (GPX) activity, and
Vigna mungo
contents of proline, ascorbic acid, total phenolic contents (TPCs) and total flavonoid con-
Vigna acontifolia
tents (TFCs) in leaves of both spp., however, catalase (CAT) exhibited varied activity. The
UV-B radiation
study concludes that substantially higher contents of TPCs and TFCs in epidermal layer,
Reactive oxygen species
proline and ascorbic acid, and higher CAT activity before and after enhanced UV-B expo-
Lipid peroxidation
sure probably attributed greater tolerance to V. acontifolia species, thus exhibited lesser UV-
Antioxidants
B induced damaging effects on cellular components and growth than that of V. mungo. This study also suggests that V. acontifolia is comparatively resistant to UV-B and thus may be useful for practical cultivation. Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.
Introduction
Though due to the successful implementation of Montreal Protocol on substances that deplete the ozone layer, there has been a reduction in incoming solar UV-B radiation (WMO,
2010), however, terrestrial ecosystems appear to be still sen, sitive due to the variations in UV-B irradiance (Ballare Caldwell, Flint, Robinson, & Bornman, 2011). Among the living organisms, plants are vulnerable to increased UV-B radiation as they are absolutely dependent on solar radiation for their survival. Several biologically active molecules such as
* Corresponding author. Tel.: þ91 5322462048, þ91 9450609911 (mobile). E-mail addresses: [email protected] (V.P. Singh), [email protected] (S.M. Prasad). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2014.12.002 1687-8507/Copyright © 2014, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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nucleic acids, proteins and lipids absorb the high energy UV-B radiation directly and get damaged (Landry, Chapple, & Last, 1995). The findings have shown that UV-B radiation can affect growth and physiological processes in plants (Musil, Chimphango, & Dakora, 2002; Mishra, Srivastava, & Prasad, 2009). Reduction in growth of photoautotroph may occur directly due to the effects of UV-B on various cell components or indirectly through enhanced generation of reactive oxygen species (ROS). The photosynthetic and respiratory electron transport systems, photorespiratory pathway and plasma membrane have been considered as potential sources of ROS and oxidative burst (Asada, 1999). ROS play various roles in cellular system that may be positive and related to the regulation of cell growth, intercellular signaling and synthesis of biologically important compounds (Mahalingam & Fedoroff, 2003). However, at elevated concentration, ROS can be extremely harmful to organisms (Jordan, 1996). Superoxide radical (O2e) is short lived and moderately reactive ROS which is readily dismutated to relatively long-lived hydrogen peroxide (H2O2). Hydrogen peroxide may diffuse some distance from its production site and at elevated concentration inhibits photosynthesis, and thus its scavenging is vital for the proper functioning of chloroplast (Foyer & Lelandais, 1996). Superoxide and H2O2 at physiological concentration do not produce negative effects, however, their toxicity arises due to formation of metal ion dependent production of hydroxyl radicals (OH), which are capable of mutating DNA and initiating chain reaction of lipid peroxidation leading to loss of function and tissue destruction (Alscher, Erturk, & Heath, 2002). To mitigate harmful effects of ROS, the aerobic organisms have been equipped with fine regulatory mechanisms to control their levels under limit. The antioxidant defense systems successfully scavenge ROS and protect cells from oxidative damage. The enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT) and peroxidases (PODs) are efficient scavengers of O2e and H2O2. The enzyme SOD is present in all sub-cellular components susceptible to oxidative stress and determines the concentration of O2e and H2O2, and is therefore called first line of defense against ROS (Alscher et al., 2002). The scavenging of H2O2 is performed by CAT and a number of PODs. CAT decomposes H2O2 to water and molecular oxygen without consuming reductant, thus provides cells with an efficient mechanism to remove H2O2 (Scandalios, 1994). PODs are monomeric haemoproteins that catalyze the oxidation of a range of substrates by H2O2. In addition to enzymatic antioxidants, organism also contains an important array of non-enzymatic antioxidants i.e. ascorbic acid, proline, phenols, flavonoids, glutathione etc. c ik, Klejdus, Stork, (Buettner & Jurkiewiez, 1996; Kova & ovska , 2011). Malc In recent time, gradual change in environment such as enhanced UV-B radiation on the Earth's surface has become an unavoidable fact and thus, causing real threat to the existing organisms. The chlorofluorocarbons, which can deplete the ozone layer and can remain in the upper atmosphere for 40e150 years, hence, the global UV-B radiation will not recover to the levels of the pre-industrialization era by the 2050, even if all the nations implement the Montreal Protocol.
Furthermore, it is known that organisms may show differential responses to the stress factors which can be explained on the basis of their morphological, genetical, biochemical and physiological features. In the present study, an attempt has been made to understand the differential responses of growth, oxidative stress and antioxidants system in two species of Vigna i.e. Vigna mungo and Vigna acontifolia at their early stage of growth against enhanced UV-B radiation. The study is significant because (i) the crop is important in maintaining the nitrogen economy of agricultural fields and nutrient value in vegetarian diet due to high protein content, and also (ii) the early stage of seedlings that play a decisive role in the crop yield being used as vegetables and pulses appears to be most vulnerable stage of growth to enhanced UV-B radiation.
2.
Materials and methods
2.1.
Plant materials and growth conditions
The seeds of V. mungo (L.) cv. IPU- 94 -1 and V. acontifolia (Jacq.) cv. RM 570 were obtained from Indian Institute of Pulses Research (IIPR), Kanpur and Regional Research Centre, Jaipur, India, respectively. Healthy seeds were surface sterilized in sodium hypochlorite solution for 15 min and washed thoroughly with sterilized double distilled water. Thereafter, seeds were soaked for 2 h in distilled water, wrapped in moistened cotton cloth and left overnight in dark for germination. The germinated seeds were sown in plastic trays containing acid washed sterilized sand and incubated in dark at 28 ± 2 C for a day. The seedlings were grown in a growth chamber at 28 ± 2 C under 13:11h light and dark periods (550 mmol photons m2 s1, PAR) with relative humidity of 60e80%. The seedlings were watered daily with double distilled water. After 3 days of growth, equal sized seedlings were gently transferred in 0.2 strength Rorison nutrient medium (pH 7.5). The nutrient medium was aerated intermittently with sterile air to avoid the anaerobic condition around roots.
2.2.
UV-B treatment
After acclimatization in nutrient medium for two days, the seedlings were given two successive exposures of UV-B on 6th and 7th day. UV-B radiation was provided by fluorescent UV-B tube (TL e 40 W/12, Philips, Holland) with its main output at 312 nm together with white light (550 mmol photons m2 s1, PAR). The UV-B tube was covered with 0.127 mm cellulose diacetate filters (Johnston Industrial plastics, Toronto, Canada to remove radiation below 280 nm) for enhanced UV-B (eUV-B, ambient þ supplemental UV-B) radiation. Cellulose diacetate filter was changed regularly to avoid aging effects on the spectral transmission of UV-B. The UV-B irradiance at the top of the plant under the tube was measured with Power Meter (Spectra Physics, Model 407, A-2, USA). At study place, the ambient UV-B radiation was 8.6 kJ m2 d1 on the summer solstice weighted against generalized plant response action spectrum. The plants beneath cellulose diacetate film received different levels of biologically effective UV-B radiation (UV-BBE) i.e. ambientþ1.2 kJ m2, ambientþ2.4 kJ m2, ambientþ3.6 kJ m2, ambientþ4.8 kJ m2 and
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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ambientþ7.2 kJ m2 simulating 4, 5, 6, 7 and 8%, respectively reduction in stratospheric ozone at Allahabad (20 470 N) during clear sky condition on the summer solstice. The various levels of enhanced UV-B radiation were achieved by managing distance between plant canopy and surface of UV-B tube. On 8th day seedlings of each set was harvested and various parameters related to growth, oxidative stress and antioxidant system were analyzed. The seedlings which were not exposed to UV-B regarded as “control”.
2.3.
Measurement of growth and relative water content
After 24 h of last UV-B exposure (on 8th day), seedlings of each set were harvested and plant fresh and dry mass was determined by single pan electronic balance (Contech- CA 223, India). For dry mass measurement, the seedlings were wrapped in butter paper and dried in an oven at 80 C for 24 h. The leaf area of treated and untreated seedlings was determined by leaf area meter (Model 211, Systronics, India). Relative water content (RWC) of leaf was determined as (FW DW)/ (TW DW) 100, where FW is the fresh weight, DW is the dry weight and TW is the turgid weight of the leaf after equilibration in distilled water for 24 h.
2.4.
Estimation of reactive oxygen species
Superoxide radical O2e was measured by the method of Elstner and Heupel (1976). This assay is based on formation of nitrite (NO 2 ) formation from hydroxylamine in the presence of O2e. The absorbance of the colored aqueous phase was recorded at 530 nm. A standard curve prepared with NaNO 2 was used to calculate the production rate of O2e. Hydrogen peroxide (H2O2) content in the treated and untreated samples was estimated by following ferrithiocyanate method as described by Sagisaka (1976). The absorbance of the solution was recorded at 480 nm, and amount of H2O2 in each sample was calculated from standard curve. Hydroxyl radical (OH) was quantified by the method of Babbs, Ann Pham, and Coolbaugh (1989), using dimethyl sulfoxide (DMSO) as molecular probe which is oxidized by hydroxyl radical into methane sulfinic acid (MSA), a stable compound. The absorbance of samples was recorded at 425 nm and an extinction coefficient 2088 M1 cm1 was used to calculate OH radicals and were expressed as MSA content.
2.5.
Estimation of oxidative damage indices
Lipid peroxidation was estimated as malondialdehyde (MDA) content adopting the method of Hodges, De Long, Forney, and Prange (1999). Malondialdehyde content was calculated using the equation: MDA (nmol ml1) ¼ (AB/157,000) 106 where A ¼ [(A532 þ TBA) (A600 þ TBA) (A532 TBA A600 TBA)] and B ¼ [(A440 þ TBA A600 þ TBA)0.0571]. Intactness of plasma membrane in leaves was measured as the leakage percentage of electrolytes as described by Gong, Li, and Chen (1998). The leakage percentage of electrolytes was calculated by using the formula (EC1/EC2) 100.
2.6.
3
Assay of enzymatic antioxidants
Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by measuring the inhibition of the reduction of p-nitroblue tetrazolium chloride (NBT) by the method of Giannopolitis and Ries (1977). The initial rate of reaction as measured by the difference in absorbance at 560 nm, in the presence and absence of extract was proportional to the amount of enzyme. One unit of SOD was defined as the amount of enzyme that inhibited NBT reduction by 50% under the specified conditions. Guaiacol peroxidase (GPX, EC 1.11.1.7) activity in leaf homogenates of treated and untreated seedlings was determined according to the method of Zhang (1992). One unit of the GPX activity is the amount of enzyme oxidizing 1 nmol guaiacol min1 at 28 C. Catalase (CAT, EC 1.11.1.6) activity was determined by following the method of Aebi (1984). One unit enzyme activity is represented as 1 mmol H2O2 decomposed min1. Protein content in each sample was estimated according to Bradford (1976).
2.7.
Estimation of non-enzymatic antioxidants
Total ascorbate was determined by the method of Gossett, Millhollon, and Cran (1994). This assay is based on reduction of Fe3þ into Fe2þ with ascorbic acid in acid solution followed by the formation of red chelate between Fe2þ and 2,2-bipyridyl. Ascorbate content was calculated by using standard curve prepared with L-ascorbic acid. Proline content in leaf homogenate of UV-B treated and untreated seedlings was estimated according to the method of Bates, Waldren, and Teare (1973). The proline content in each sample was calculated from the standard curve. Total phenolic contents (TPCs) were estimated following the method of Waterhouse (2001). The amount of TPCs was expressed as mg gallic acid equivalents g1 dry weight. Total flavonoid contents (TFCs) in treated and untreated seedlings were determined spectrophotometrically using AlCl3 method using quercetin as standard (Zhishen, Mengcheng, & Jianming, 1999). After incubating reaction mixtures at room temperature, optical density was measured at 512 nm. TFCs were expressed as mg quercetin equivalents g1 dry weight.
2.8.
Statistical analysis
Results were statistically analyzed by analysis of variance (ANOVA). Duncan's multiple range tests was applied for mean separation for significant differences among treatments using SPSS software (Version 10, SPSS Inc., Chicago). Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6).
3.
Results
3.1.
Growth
UV-B radiation induced impact on growth of V. mungo and V. acontifolia seedlings was analyzed and results are presented in
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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Table 1 e Effect of enhanced UV-B radiation on plant fresh mass (PFM, mg plant¡1), plant dry mass (PDM, mg plant¡1) and relative water content (RWC) of Vigna mungo and Vigna acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent decrease (¡) over control value. UV-B exposure
Growth parameters Vigna mungo PFM
Control Ambþ1.2 kJ Ambþ2.4 kJ Ambþ3.6 kJ Ambþ4.8 kJ Ambþ7.2 kJ
2
m m2 m2 m2 m2
324.0 310.0 298.0 281.6 267.3 251.4
± 5.37a ± 5.32 (4)b ± 2.70 (8)c ± 2.43 (13)d ± 3.84 (18)e ± 3.25 (22)f
Vigna acontifolia
PDM 27.2 26.1 24.8 23.1 22.1 20.8
RWC (%)
± 0.33a ± 0.37 (4)a ± 0.54 (9)b ± 0.25 (15)c ± 0.53 (19)cd ± 0.17 (24)ef
91.6a 91.3a 90.6a 90.2a 89.9a 89.8a
Tables 1 and 2. UV-B declined (P < 0.05) growth parameters: biomass accumulation in leaves and in whole plant and also on leaf area in dose dependent manner in both the species. Leaf area showed maximum reduction (P < 0.05) compared to other growth parameters which exhibited 35% decline in V. mungo and only 19% in V. acontifolia following Ambþ7.2 kJ m2 UV-B exposure. Similar UV-B dose decreased leaf dry mass by 29% and 19%, and plant dry mass by 24% and 20% in V. mungo and V. acontifolia, respectively. In contrast to growth parameters, RWC was not affected significantly (P < 0.05) by UV-B in both the species (Table 1).
3.2.
3.3.
PFM 206.6 201.2 193.7 186.7 179.4 172.0
± 4.0g ± 5.31 ± 4.44 ± 2.94 ± 2.64 ± 4.82
PDM 21.5 20.5 19.7 19.2 18.4 17.3
gh
(3) (6)hi (10)ij (13)jk (17)k
RWC (%)
± 0.32de ± 0.53 (5)ef ± 0.38 (8)fg ± 0.44 (11)gh ± 0.33 (14)hi ± 0.29 (20)i
89.8a 89.6a 89.5a 89.1a 88.8a 88.6a
Lipid peroxidation and membrane damage
The data presented in Fig. 2a reveal that UV-B radiation caused substantial damage to cellular membrane in both species as malondialdehyde (MDA) content, a peroxidation product of lipid raised progressively following the exposure of leaves with increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) of enhanced UV-B radiation. The accumulation of MDA content was significantly (P < 0.05) higher in V. mungo, and after Ambþ7.2 kJ m2 of UV-B exposure MDA content was enhanced by 119% and 81% in V. mungo and V. acontifolia, respectively over their respective control. Further, the oxidative stress induced damaging effect was also analyzed by determining the electrolyte leakage in the leaves of both the species. The results presented in Fig. 2b depicts that UV-B exposure with increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) caused progressive increase in electrolyte leakage in leaf tissues; however, the damaging effect was stronger (P < 0.05) in V. mungo than V. acontifolia.
Reactive oxygen species
The results pertaining to superoxide radical (O2e), hydrogen peroxide (H2O2) and hydroxyl radical (OH) contents in the leaves of UV-B exposed seedlings are depicted in Fig. 1aec. Compared to the V. acontifolia, the control seedlings of V. mungo exhibited significantly (P < 0.05) higher contents of O2e, H2O2 and OH showing 180%, 12% and 21%, respectively higher amounts than the respective values in V. acontifolia. UV-B dose (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) dependent rise in ROS contents was noticed in both species but the rate of generation/accumulation of O2e, H2O2 and OH was substantially higher in V. mungo.
3.4.
Enzymatic antioxidants
The results pertaining to superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) are shown in Table 3.
Table 2 e Effect of enhanced UV-B radiation on leaf area (LA, mm2 plant¡1), leaf fresh mass (LFM, mg plant¡1) and leaf dry mass (LDM, mg plant¡1) of Vigna mungo and Vigna acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent decrease (¡) over control value. UV-B exposure
Growth parameters Vigna mungo LA
Control Ambþ1.2 kJ Ambþ2.4 kJ Ambþ3.6 kJ Ambþ4.8 kJ Ambþ7.2 kJ
m2 m2 m2 m2 m2
348.1 329.4 317.8 274.0 254.4 227.1
± 4.46a ± 4.73 (5)b ± 4.91 (9)b ± 4.44 (21)e ± 3.57 (27)g ± 3.60 (35)h
LFM 56.4 ± 54.6 ± 52.5 ± 47.9 ± 41.3 ± 37.2 ±
0.43a 0.36 (3)b 0.32 (7)c 0.17 (15)d 0.45 (27)g 0.32 (34)i
Vigna acontifolia LDM 7.8 ± 0.19a 7.3 ± 0.18 (6)b 6.7 ± 0.20 (14)c 6.4 ± 0.19 (18)cd 6.0 ± 0.13 (23)def 5.5 ± 0.17 (29)fg
LA 319.7 ± 304.2 ± 295.6 ± 290.4 ± 269.8 ± 257.4 ±
3.16b 5.21 (5)c 4.83 (8)cd 4.84 (9)d 3.89 (16)ef 4.29 (19)fg
LFM 44.2 42.7 41.5 40.8 38.4 36.6
± 0.41e ± 0.51 (3)f ± 0.43 (6)g ± 0.23 (8)g ± 0.23 (13)h ± 0.26 (17)i
LDM 6.7 ± 6.5 ± 6.3 ± 6.1 ± 5.8 ± 5.4 ±
0.19c 0.15 (3)cd 0.18 (6)cde 0.15 (9)de 0.17 (13)efg 0.15 (19)g
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Ambþ7.2 kJ m2), however, it was maximum in leaves of V. mungo and V. acontifolia after Ambþ2.4 kJ m2 and Ambþ4.8 kJ m2 of UV-B exposure, respectively. CAT activity in V. mungo initially (up to Ambþ2.4 kJ m2 of UV-B exposure) showed increasing trend and thereafter, it declined progressively exhibiting about 14% less activity over the value of control when leaves were irradiated with Ambþ7.2 kJ m2. In V. acontifolia CAT activity though exhibited a declining trend following the increasing UV-B (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2) exposure but the activity was still higher (P < 0.05) in comparison to the V. mungo. UV-B exposure caused an accelerating effect on GPX activity in both the species and increased progressively in V. mungo while it was maximum in Ambþ4.8 kJ m2 UV-B treated leaves of V. acontifolia.
3.5.
Non-enzymatic antioxidants
The results pertaining to ascorbic acid, proline, total phenolic contents (TPCs) and total flavonoid contents (TFCs) in leaves of both the species are depicted in Fig. 3aed, respectively. The untreated leaves of V. acontifolia exhibited comparatively higher contents of non-enzymatic antioxidants showing 41%, 43%, 44% and 61% more ascorbic acid, proline, TPCs and TFCs than V. mungo. The increasing doses (Ambþ1.2 kJ m2 to Ambþ7.2 kJ m2 min) of UV-B caused progressive rise in ascorbic acid and proline contents however, a steep decline (P < 0.05) in V. mungo was recorded following Ambþ7.2 kJ m2 of UV-B exposure. There was significant enhancement in TPCs and TFCs in V. mungo with the rising doses of UV-B. In V. acontifolia TFCs showed initially increasing trend (up to Ambþ2.4 kJ m2 of UV-B exposure) which was substantially high. Further, increase in UV-B dose (Ambþ3.6 kJ m2 to Ambþ7.2 kJ m2) caused a declining trend; however, the content was significantly (P < 0.05) higher compared to the values obtained in V. mungo under similar condition.
4.
Fig. 1 e Superoxide radical (a), hydrogen peroxide (b) and hydroxyl radical (c) contents in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test.
GPX activity was significantly higher in the leaves of control V. mungo leaves while CAT activity was appreciably more in V. acontifolia. SOD activity in both species exhibited an enhanced rate following UV-B exposure (Ambþ1.2 kJ m2 to
Discussion
UV-B dose dependent reduction in growth parameter (biomass accumulation) in V. mungo and V. acontifolia seedlings was recorded (Tables 1 and 2). Besides this, visual symptoms such as dwarfing of plants, distortion of shoots, and reduction in leaf dimension in the seedlings of both the species were also observed (data not shown). Similar to our results enhanced level of UV-B radiation significantly declined the growth in Vigna unguiculata (Musil et al., 2002). Such reduction in growth could be associated with UV-B induced inhibition in photosynthetic rate and destruction of growth promoting hormone: indole acetic acid (IAA) (Kulandaivelu, Maragatham, & Nedunchezhian, 1989). Further, it can also be linked with excessive generation/accumulation of free radicals (Fig. 1aec) that cause damaging effect on structural and functional components of cells. UV-B induced strong reduction in growth of Vigna species at higher dose particularly in V. mungo might have occurred due to irreparable damage to biomolecules associated with photosynthetic apparatus particularly to the membrane involved in energy transducing system and also to the C3 cycle enzymes as
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
2.33h 2.82 (þ8)h 2.67 (þ13)h 4.64 (þ50)f 3.39 (þ108)d 4.01 (þ33)g 85.6 ± 92.6 ± 96.3 ± 128.3 ± 178.1 ± 114.0 ± 28.1 ± 0.54a 25.8 ± 0.57 (8)b 23.6 ± 0.58 (16)c 21.8 ± 0.58 (22)d 19.5 ± 0.32 (31)e 17.7 ± 0.60 (37)f ± 0.16f ± 0.15 (þ5)f ± 0.15 (þ13)ef ± 0.13 (þ25)e ± 0.19 (þ28)e ± 0.15 (þ5)f 4.0 4.2 4.5 5.0 5.1 4.2 3.0g 4.07 (þ30)e 6.07 (þ90)c 6.05 (þ125)b 4.17 (þ134)ab 3.29 (þ140)a 114.8 ± 149.1 ± 218.2 ± 257.8 ± 269.2 ± 275.0 ± ± 0.65gh ± 0.50 (þ8)fg ± 0.33 (þ14)f ± 0.39 (þ8)fg ± 0.41 (8)hi ± 0.61 (14)i 15.3 16.5 17.5 16.5 14.1 13.1 0.44d 0.39 (þ28)c 0.19 (þ62)a 0.17 (þ43)b 0.31 (þ40)b 0.34 (þ24)c 10.5 ± 13.4 ± 17.0 ± 15.0 ± 14.7 ± 13.0 ± kJ m2 kJ m2 kJ m2 kJ m2 kJ m2 Control Ambþ1.2 Ambþ2.4 Ambþ3.6 Ambþ4.8 Ambþ7.2
CAT (units mg1 protein min1)
V. acontifolia
SOD (units mg1 protein) CAT (units mg1 protein min1)
V. mungo
GPX (units mg1 protein min1)
Enzyme activities
SOD (units mg1 protein)
observed in earlier findings (Bischof, Hanelt, & Wiencke, 2000; Mishra, Srivastava, Prasad, & Abraham, 2008). UV-B exposure may stimulate the generation of ROS in plants by disrupting electron transport system (ETS) and diverting the normal path of electron in the photosynthetic and respiratory electron transport systems (Halliwell & Gutteridge, 1999). Disruption of ETS in various cellular compartments resulted in enhanced generation/accumulation of ROS thus, O2e, H2O2 and OH, the potent inhibitors of a number of key metabolic processes, increased substantially in UV-B exposed Vigna species seedlings (Fig. 1aec). Compared to the V. acontifolia, the contents of O2e, H2O2 and OH were found to be appreciably higher in V. mungo control leaves and the amount of ROS increased continuously with rising doses of UV-B. The greater accumulation of ROS particularly H2O2 in V. mungo leaves could be correlated with UV-B induced substantial damaging effects on photosynthetic electron transport and CO2 fixation as reported in V. unguiculata (Mishra
UV-B exposure
Fig. 2 e Lipid peroxidation as MDA content (a) and electrolyte leakage (b) in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test.
GPX (units mg1 protein min1)
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Table 3 e Effect of enhanced UV-B radiation on antioxidant enzyme activities in leaves of V. mungo and V. acontifolia seedlings. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). Different superscripts denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Data in parentheses denote per cent increase (þ) or decrease (¡) over control value.
6
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et al., 2008). Thus, this condition probably led to the spilling of electron to O2 resulting in the enhanced generation of ROS. ROS produce damaging effects on functional biomolecules and if there were excess ROS formed within the cells, one would expect impairment in several metabolic processes that eventually lead to cell death. H2O2 at elevated level (mM concentration) is known to inactivate enzymes by oxidizing their thiol groups as reported in case of Calvin cycle enzymes (Devine, Duke, & Fedtke, 1993, ). However, the real threat of O2e and H2O2 is their potential to act as precursors for OH as we have observed increased amount of OH in UV-B dose dependent manner (Fig. 1c). The OH can readily oxidize amino acid residues of proteins, fatty acids of phospholipids and deoxyribose and bases in DNA (Halliwell & Gutteridge, 1999). In the present study, even untreated seedlings of two Vigna species showed significant (P < 0.05) differences in MDA content (Fig. 2a) which could be linked with faster rate of physiological activity in V. mungo (Tables 1 and 2) leading to more generation/accumulation of O2e, H2O2 and OH (Fig. 1aec). Further, UV-B exposure increased the MDA content in both the species of Vigna in UV-B dose dependent manner
7
and it was significantly (P < 0.05) higher in UV-B exposed V. mungo leaves compared to V. acontifolia (Fig. 2a). The membrane stability of leaf discs of Vigna species seedlings also showed similar trend as observed in MDA content (Fig 2b). Lower lipid peroxidation and lower electrolyte leakage in V. acontifolia seedlings may be associated with relative tolerance of this species to UV-B, while the reverse was noticed for V. mungo seedlings. To mitigate the negative impact of oxidative stress, plants have efficient antioxidative defense system which provides adequate protection against ROS and free radicals. However, under severe stress condition the antioxidant capacity of plants may not bring the level of ROS under limit thus, oxidative injury takes place. The antioxidant enzymes induced by UV-B are likely to be species specific. Activities of SOD, CAT, APX and GPX were reported to be enhanced by UV-B radiation (Kondo & Kawashima, 2000; Mishra et al., 2009) while decreased CAT activity was reported in barley seedlings (Zancan, Suglia, La Rocca, & Ghisi, 2008). In the present study, the response of antioxidant enzymes: SOD, CAT and GPX varied with UV-B exposure and species tested (Table 3).
Fig. 3 e Ascorbic acid (a), proline (b), total phenolic contents (c) and total flavonoid contents (d) in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation. Values are mean ± standard error of three independent experiments with the two replicates in each experiment (n ¼ 6). The bars with different letters denote significance at P < 0.05 between the species for a given treatment according to the Duncan's multiple range test. Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
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Though the leaves of V. mungo seedlings exhibited higher rate of SOD and GPX activities which showed increasing trends with UV-B dose (except Ambþ7.2 kJ m2), however, there was considerable accumulation of ROS leading to enhanced damaging effects (Fig. 2a and b). The greater accumulation of the ROS (Fig. 1aec) in V. mungo probably resulted due to the imbalance between generation/accumulation of ROS and antioxidant systems. In comparison to the V. acontifolia, V. mungo before and after UV-B exposure exhibited lower CAT activity that might be partially responsible for higher accumulation of ROS (H2O2 and OH). The substantial decrease in CAT activity at high UV-B doses might have occurred either due to down regulation of the genes coding for this enzyme or ROS mediated damaging effect. In the present study, under UV-B stress, there was substantial increase in GPX activity in V. mungo seedlings (Table 3). The accelerated GPX activity can also be correlated with reduced growth and biomass accumulation as peroxidase may initiate the catabolism of growth promoting hormone like indole acetic acid (IAA) (Kulandaivelu et al., 1989), which is supported by the increased UV-B sensitivity of tobacco transgenic plants with decreased auxin levels (Lagrimini, 1999). Apart from enzymatic antioxidants, plant cells also contain an important array of non-enzymatic antioxidants such as ascorbic acid, proline, phenolic compounds, flavonoids, glutathione etc. for mitigating the toxic effects of ROS. The low molecular weight antioxidants are even helpful in scavenging those ROS (singlet oxygen; 1O2 and OH) for which no enzymatic scavenging system has been evolved. Ascorbic acid is one of the most powerful antioxidants that scavenges the H2O2 and other ROS profoundly. Protective effect of ascorbic acid is possibly more related to its participation in direct scavenging of 1O2 and OH, and also the removal of H2O2 through Asada-Halliwell pathway thus, reducing ROS induced damage to essential proteins and/or nucleic acids (Buettner & Jurkiewiez, 1996). Significantly (P < 0.05) higher ascorbic acid content in V. acontifolia leaves before (in control plants) and after UV-B exposure showed its greater capability to scavenge ROS (Fig. 3a). There may be two possibilities regarding increased ascorbic acid content; either its synthesis has been amplified or its regeneration through Asada-Halliwell pathway. Proline content has also been shown to be increased in plants exposed to various stresses including UV-B (Mahdavian, Ghorbanli, & Kalantari, 2008) and similar trend was also recorded in the present study (Fig. 3b). Further, significantly (P < 0.05) higher proline content in V. acontifolia justified its protective role against UV-B stress as noticed in earlier finding (Mahdavian et al., 2008). The phenols and flavonoids (UV-B absorbing pigments) provide the first line of defense against UV-B as they are primarily located in leaf epidermal layers, preventing its penetration to the leaf interior. The importance of flavonoids in leaf epidermal layers was confirmed by applying a fiber-optic c ik microprobe (Bornman & Vogelmann, 1991). Further, Kova et al. (2011) have reported importance of TPCs and TFCs in UV protection in a lichen- Xanthoria parietina. Compared to the V. mungo, significantly (P < 0.05) higher TPCs and TFCs in V. acontifolia leaves before and after UV-B exposure probably prevented the penetration of UV-B radiation to the leaf
interior, thus suggesting better adaptability towards UV-B stress (Fig. 3c and d).
5.
Conclusion
The study concludes that enhanced UV-B exposure caused significant reduction in growth in both the Vigna species; however, the damaging effects were more prominent in V. mungo. In spite of high SOD and GPX activities, the greater accumulation of ROS and associated membrane damage confirm the higher sensitivity of V. mungo seedlings to enhanced UV-B irradiation. Appreciably higher contents of ascorbic acid, proline, TPCs and TFCs, and comparatively more activity of CAT in V. acontifolia leaves before and after UV-B exposure render it as a more resistant species towards enhanced UV-B while the reverse is apparently true for V. mungo.
Acknowledgments Rajiv Dwivedi and Vijay Pratap Singh are thankful to CSIR and UGC, respectively for providing financial support in the form of JRF and SRF to carry out this work. The financial assistance provide to “Jitendra Kumar” as JRF under the scheme RGNF2012-13-SC-UTT-33185 funded by UGC New Delhi, India.
references
Aebi, I. I. (1984). Catalase in vitro. Methods Enzymology, 105, 121e126. Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutase (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 53, 1331e1341. Asada, K. (1999). The water - water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology Plant Molecular Biology, 50, 601e639. Babbs, C. F., Ann Pham, J., & Coolbaugh, R. C. (1989). Lethal hydroxyl radical production in paraquat-treated plants. Plant Physiology, 90, 1267e1270. , C. L., Caldwell, M. M., Flint, S. D., Robinson, S. A., & Ballare Bornman, J. F. (2011). Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change. Photochemistry and Photobiology Science, 10, 226e241. Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid determination of free proline in water-stress studies. Plant and Soil, 39, 205e207. Bischof, K., Hanelt, D., & Wiencke, C. (2000). Effect of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae. Planta, 211, 555e562. Bornman, J. F., & Vogelmann, T. C. (1991). Effect of UV-B radiation on leaf optical properties measured with fiber optics. Journal of Experimental Botany, 42, 547e554. Bradford, M. (1976). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Buettner, G. R., & Jurkiewiez, B. A. (1996). Chemistry and biochemistry of ascorbic acid. In E. Cadenas, & L. Packer (Eds.),
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002
J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 4 ) 1 e9
Handbook of antioxidants (pp. 91e115). New York: Marcel Dekker. Devine, M., Duke, S. O., & Fedtke, C. (1993). Physiology of herbicidal action. Englewood Cliffs: PTR Prentice-Hall. Elstner, E. F., & Heupel, A. (1976). Inhibition of nitrite formation from hydroxyl ammonium chloride a simple assay per superoxide dismutase. Analytical Biochemistry, 70, 616e620. Foyer, C. H., & Lelandais, M. A. (1996). A comparison of the relative rates of transport of ascorbate and glucose across the thylakoid, chloroplast and plasma membranes of pea leaves mesophyll cells. Journal of Plant Physiology, 148, 391e398. Giannopolitis, C. N., & Ries, S. K. (1977). Superoxide dismutase. I. Occurrence in higher plants. Plant Physiology, 59, 309e314. Gong, M., Li, Y. J., & Chen, S. Z. (1998). Abscisic acid induced thermotolerance in maize seedlings is mediated by calcium and associated with antioxidant system. Journal of Plant Physiology, 153, 488e496. Gossett, D. R., Millhollon, E. P., & Cran, L. M. (1994). Antioxidant response to NaCl stress in salt sensitive cultivars of cotton. Crop Science, 34, 706e714. Halliwell, B., & Gutteridge, J. M. C. (1999). Free radicals in biology and medicine (3rd ed.). Oxford: Oxford University Press. Hodges, D. M., De Long, J. M., Forney, C. F., & Prange, R. K. (1999). Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207, 604e611. Jordan, B. R. (1996). The effects of ultraviolet-B radiation on plants: a molecular perspective. Advance in Botanical Research, 22, 97e162. Kondo, N., & Kawashima, M. (2000). Enhancement of the tolerance to oxidative stress in cucumber (Cucumis sativus L.) seedlings by UV-B irradiation: possible involvement of phenolic compounds and antioxidative enzymes. Journal of Plant Research, 113, 311e317. c ik, J., Klejdus, B., Stork, ovska , S. (2011). Sensitivity Kova F., & Malc of Xanthoria parietina to UV-A: role of metabolic modulators. Journal of Photochemistry and Photobiology B: Biology, 103, 243e250. Kulandaivelu, G., Maragatham, S., & Nedunchezhian, N. (1989). On the possible control of ultraviolet eB induced response in growth and photosynthetic activities in higher plants. Physiologia Plantarum, 76, 398e404. Lagrimini, L. M. (1999). The role of peroxidase in auxin metabolism. In L. M. Lagrimini (Ed.), Abstracts peroxidase 99. International symposium for plant peroxidases (p. 27). Columbus: Ohio State University.
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Landry, L. C., Chapple, C. C., & Last, R. L. (1995). Arabidopsis mutants lacking phenolics sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiology, 109, 1159e1166. Mahalingam, R., & Fedoroff, N. (2003). Stress response, cell death and signaling: the many faces of active oxygen species. Physiologia Plantarum, 119, 56e68. Mahdavian, K., Ghorbanli, M., & Kalantari, K. M. (2008). The effect of UV-B radiation on the content of chlorophyll, flavonoids, anthocyanin and proline in Capsicum annuum L. Turkish Journal of Botany, 32, 25e33. Mishra, V., Srivastava, G., & Prasad, S. M. (2009). Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation. Scientia Horticulturae, 120, 373e378. Mishra, V., Srivastava, G., Prasad, S. M., & Abraham, G. (2008). Growth, photosynthetic pigments and photosynthetic activity during seedling stage of cowpea (Vigna unguiculata) in response to UV-B and dimethoate. Pesticide Biochemistry and Physiology, 92, 30e37. Musil, C. F., Chimphango, S. B. M., & Dakora, F. D. (2002). Effects of elevated ultraviolet-B radiation on native and cultivated plants of southern Africa. Annals of Botany, 90, 127e137. Sagisaka, S. (1976). The occurrence of peroxide in a perennial plant Populus gelrica. Plant Physiology, 57, 308e309. Scandalios, J. G. (1994). Regulation and properties of plant catalases. In C. H. Foye, & P. M. Mullineaux (Eds.), Causes of photooxidative stress and amelioration of defense system in plants (pp. 275e315). Boca Raton: CRC Press. W.M.O. (2010). Scientific assessment of ozone depletion: 2010. Global Ozone Research and Monitoring ProjecteReport No. 52, 516 pp. Geneva, Switzerland, 2011. Waterhouse, A. L. (2001). Determination of total phenolics, handbook of food analytical chemistry, unit I 1.1: Polyphenolics (pp. 464e465). New York: Wiley. Zancan, S., Suglia, I., La Rocca, N., & Ghisi, R. (2008). Effect of UV-B radiation on antioxidant parameters of iron-deficient barley plants. Environmental and Experimental Botany, 63, 71e79. Zhang, X. Z. (1992). The measurement and mechanism of lipid peroxidation and SOD, POD and CAT activities in biological system. In X. Z. Zhang (Ed.), Research methodology of crop physiology (pp. 208e211). Beijing: Agriculture Press. Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64, 555e559.
Please cite this article in press as: Dwivedi, R., et al., Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Sciences (2014), http://dx.doi.org/10.1016/ j.jrras.2014.12.002