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Environmental and Experimental Botany 59 (2007) 21–33

Changes in oxidative stress defense system in wheat(Triticum aestivum L.) and mung bean (Vigna radiata L.)cultivars grown with and without mineral nutrients and

irradiated by supplemental ultraviolet-B

S.B. Agrawal a,∗, Dheeraj Rathore b

a Laboratory of Air Pollution and Global Climatic Change, Department of Botany, Banaras Hindu University, Varanasi 221005, Indiab Department of Botany, Allahabad Agricultural Institute, Deemed University, Allahabad 211007, India

Received 21 April 2005; received in revised form 31 July 2005; accepted 30 September 2005

Abstract

Field study was conducted to evaluate the inter- and intra-specific variations in sensitivity of two cultivars each of wheat (Triticum aestivumL. cv. HD 2329 and HUW 234) and mung bean (Vigna radiata L. cv. Malviya Jyoti and Malviya Janpriya) to supplemental levels of UV-Birradiation (sUV-B, 280–315 nm) with and without recommended levels of mineral nutrients. Results showed decrease in photosyntheticpigments and biomass of all the four cultivars due to sUV-B radiation. Antioxidative defense system was activated in all the cultivars afterirradiation with sUV-B. SOD, peroxidase and total thiol contents increased, while catalase activity and ascorbic acid contents decreased undersUV-B irradiation. On the basis of biomass, UV-B sensitivity can be arranged in decreasing order as: Malviya Janpriya < Malviya Jyoti < HD2329 < HUW 234. Application of mineral nutrients (N, P and K) showed significant positive response in all cultivars by ameliorating thenegative impact of sUV-B.© 2005 Elsevier B.V. All rights reserved.

Keywords: Ultraviolet-B radiation; Mineral nutrients; Wheat (Triticum aestivum L.); Mung bean (Vigna radiata L.); Photosynthetic pigments; Antioxidantd

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. Introduction

Stratospheric O3 reduction is one of the pressing globaloncerns of climate change, which has prompted recentfforts in assessing the potential damage to vegetation due toupplemental levels of ultraviolet-B (sUV-B, 280–320 nm)adiation (Caldwell et al., 1998; Hollosy, 2002; Kakani etl., 2003). Numerous studies have investigated the effects oflevated UV-B on plants, and have shown a diverse range of
esponses, including changes at the physiological, morpho-ogical, biochemical and molecular levels (Caldwell et al.,995; Jordan, 1996; Allen et al., 1998; Paul, 2001).Thesetudies have shown deleterious effects of UV-B such as
∗ Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174.E-mail address: [email protected] (S.B. Agrawal).

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098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2005.09.009

educed photosynthesis, biomass reduction, decreased pro-ein synthesis, damage to nucleic acids and lipids (Jordan,996; Jansen et al., 1998; Rathore et al., 2003).

Under light conditions, photoreduction of O2 in greenlants is an unavoidable process that can results in superoxidenion (O2

•−) production. Dismutation of O2•− by superox-

de dismutase (SOD) results in the formation of oxygen andydrogen peroxide (H2O2) and the latter can react with O2

•−o create the highly reactive hydroxyl radical (•OH) via theaber–Weiss cycle (Bowler et al., 1992). Different speciesf plants accomplish protection towards stress through dif-erent biochemical adjustments but reactive oxygen speciesROS) scavenging is a common response to most stresses.
OS scavenging depends on the detoxification mechanismrovided by an integrated system of nonenzymatic reducedolecules like ascorbate and glutathione as well as enzy-atic antioxidants like SOD, catalase and peroxidase (Dai

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t al., 1997; Srivalli et al., 2003). At present, our knowledgeoncerning the role of the antioxidant system in protectinglants under UV-B stress is limited because few studies haveeen made covering a small number of plant species (Costat al., 2002; Kakani et al., 2003).

Responses of plants to UV-B vary not only among thepecies, but also among the cultivars of same species (Tevini,000). Variations in responsiveness of different species andultivars to UV-B were also reported for a variety of plantpecies (Dai et al., 1994; Smith et al., 2000; Alexieva et al.,001; Yanqun et al., 2003a,b; Zu et al., 2004). The effects
f UV-B on leaves can be mimicked by free radical genera-ors and prevented by antioxidant feeding (Mackerness et al.,998). Smith et al. (2000) concluded that variations in UV-
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ig. 1. Age wise changes in total chlorophyll content of control and sUV-B exposecv. Malviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (bars

Experimental Botany 59 (2007) 21–33

sensitivity between different species represent the relativeontribution of morphological, physiological and biochemi-al differences, but variations within species are usually moreubtle.

Besides inter and intraspecific variations in UV-B sen-itivity, other abiotic factors also alter and/or modify thelant responses as an outcome of the interactions. Under fieldonditions, plants usually experience several stresses simul-aneously. The effect of enhanced UV-B radiation on plantsan be modified by other co-occurring stresses or by simplyhanging environmental factors like atmospheric CO (Bjorn
2t al., 1997), water availability (Manetas et al., 1997) andutrient availability (Murali and Teramura, 1987; Levizound Manetas, 2001).
d Triticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata Lrepresent ± 1S.E.).

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Most studies have examined the responses of individualpecies and/or their varieties to UV-B grown under unrealisticnd unbalanced UV-B, UV-A and photosynthetically activeadiation (Caldwell et al., 1995; Middleton and Teramura,994) in growth chambers and greenhouses (Fiscus andooker, 1995), or under balanced UV-B radiation in the field,ut at diverse locations with dissimilar environmental con-itions (Rozema et al., 1997). Therefore, the present studyas conducted in the field under the natural level of PAR

t two different doses of UV-B irradiation i.e. ambient andmbient + supplemental (7.1 kJ m−2, simulating 20% ozoneepletion at Allahabad, 20◦47′N) on two cultivars each ofheat and mung bean with and without the recommended

evel of mineral nutrients to evaluate its effect on photosyn-hetic pigment, biomass and antioxidative response amongultivars. We hypothesized that sUV-B radiation leads toroduction of ROS, which causes reduction in photosyn-hetic pigments and also affect several biochemical processes.nsufficient removal of ROS by ascorbic acid, SOD and thiolsill affect various metabolic processes, causing a reduction iniomass. Further, plants may develop resistance against sUV-irradiation by nutrient’s application to mitigate its negative

mpact via more activation of the antioxidant defense system.

. Materials and methods

.1. Plant materials and growth conditions

The study was performed at the agricultural farm ofllahabad Agricultural Institute of Allahabad city of state

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able 1-ratios and levels of significance of three way ANOVA test for pigments and enzymadiata L. (cv Malviya Jyoti and Malviya Janpriya) plants

ultivars Parameters Factor

Plant age (A) UV-B treatment (T)

D 2329 Total chlorophyll 36.36*** 13.37***

Carotenoids 22.78*** 8.12**

Ascorbic acid 1034.3*** 40.35***

Thiol 14.92*** 68.04***

UW 234 Total chlorophyll 33.78*** 14.74***

Carotenoids 48.02*** 109.52***

Ascorbic acid 1314.4*** 18.03***

Thiol 26.02*** 61.16***

alviya Jyoti Total chlorophyll 31.01*** 14.12***

Carotenoids 6.39* 1.44NS

Ascorbic acid 238.55*** 23.47***

Thiol 0.62NS 24.19***

alviya Janpriya Total chlorophyll 6.34** 2.78NS

Carotenoids 44.22*** 35.71***

Ascorbic acid 269.28*** 137.69***

Thiol 2.77NS 10.09**

S: not significant.* Significant at p < 0.05.

** Significant at p < 0.01.** Significant at p < 0.001.

Experimental Botany 59 (2007) 21–33 23

ttar Pradesh, Eastern Gangetic plains of India situated at4◦47′N latitude and 82◦21′E longitude, and 96 m aboveean sea level. Soil type was alluvial with pH 7.62,

rganic carbon 1.64%, N 690 mg 100 g−1 soil, P 16.4 mg00 g−1 soil and K 136.2 mg 100 g−1 soil. Genetically sim-lar seeds of two cultivars each of wheat (Triticum aes-ivum L. cv. HD 2329 and HUW 234) and mung beanVigna radiata L. cv. Malviya Jyoti and Malviya Janpriya)ere sown separately under field conditions in 24 plots of

ize 1 m × 1 m in respective growing seasons. After ger-ination, plants were thinned in each row (spaced 30 cm

part) for uniformity in growth to one plant every 15 cm.lants were watered uniformly. Four border rows wereown round each plot in order to minimize heterogeneity inicroclimate.

.2. Experimental design and nutrient application

The experimental design was a split plot with UV-B treat-ents as whole plots and fertilizer treatments as the sub plots

andomized within the whole plots. Each treatment was repli-ated three times. The experiment had three factors (i) sUV-Breatment, (ii) fertilizer treatments and (iii) plant age. Effectsf all the three factors were studied singly and in combina-ion. The different fertilizer treatments were: recommendedose of nutrients (F1), and without nutrients application (F0).he recommended dose (RD) of N, P and K for wheat were
0, 40 and 40 kg ha−1, respectively and for mung bean 20 and0 kg ha−1 of N and P, respectively. N, P and K were applieds urea, single superphosphate and muriate of potash, respec-ively. A half dose of N and full doses of P and K were given
e activity of Triticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna

Fertilizer dose (F) A × T A × F T × F A × T × F

100.54*** 1.12NS 8.85*** 0.33NS 0.04NS

31.49*** 0.38NS 1.29NS 00NS 0.01NS

89.47*** 0.12NS 10.82*** 2.63NS 0.24NS

45.62*** 2.32NS 0.46NS 1.39NS 0.01NS

31.6*** 1.46NS 2.04NS 0.03NS 0.01NS

392.0*** 4.34* 0.86NS 0.05NS 0.04NS

72.13*** 1.43NS 4.76* 0.33NS 0.27NS

46.59*** 0.8NS 4.92* 1.14NS 0.01NS

20.95*** 0.38NS 1.35NS 0.12NS 0.01NS

7.45* 0.09NS 0.61NS 0.05NS 0.01NS

40.78*** 1.67NS 2.28NS 2.70NS 0.16NS

7.30* 0.01NS 0.14NS 0.72NS 0.01NS

13.27*** 0.13NS 0.07NS 0.32NS 0.02NS

321.41*** 0.56NS 2.48NS 0.8NS 0.07NS

96.30*** 4.61* 1.91NS 6.05* 0.88NS

1.12NS 0.01NS 0.01NS 0.17NS 0.01NS

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s basal dose and another half dose of N was given as a topressing.

.3. sUV-B treatment

sUV-B was artificially provided by Q Panel UV-B 313uorescent lamps (Q Panel, Cleveland, Ohio, USA). Banksf 4 lamps (120 cm long) fitted 30 cm apart on a steel frameere suspended above and perpendicular to the planted rowsf each plots. The 30 cm distance between the top of thelant canopy and UV-B lamps was kept constant. UV-Breatment was started just after germination for 5 h day−1

n the middle of the photoperiod till the maturity. The UV-irradiance at the top of the plant canopy under the lamps

as measured with an ultraviolet intensity meter (UVP Inc.,an Gabriel, USA). The readings were converted to UV-

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ig. 2. Age wise changes in carotenoid content of control and sUV-B exposed Trialviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (bars repr


BE values by comparing with the Spectro Power MeterScientech, Boulder, USA). Plants under 0.13 mm polyesterlter lamps received only ambient UV-B (8.6 kJ m−2 UV-BE) on the summer solstice weighted against generalizedlant response action spectrum of Caldwell (1971). Thelants beneath 0.13 mm cellulose diacetate film received UV-BE (ambient + 7.1 kJ m−2) that mimicked 20% reduction in

tratospheric ozone at Allahabad (20◦47′N) during clear skyondition on the summer solstice (Green et al., 1980) nor-alized at 300 m. Cellulose diacetate and polyster films used

o transmit UV-B (cut off ca. 292 nm) and exclude UV-B (cutff ca. 318 nm). The ozone column thickness was assumed at
.0 mm, the albedo 0 and the scatter 1.0. Filters were changedrequently to avoid aging effects on the spectral transmissionf UV-B. For convenience, control plants were designated as0 for those grown without fertilizer, F1 grown at the rec-
ticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv.esent ± 1S.E.).

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mmended dose of fertilizer, with the corresponding sUV-Breated plants designated as F0T, F1T.

.4. Plant Analysis

Wheat plants were sampled in triplicate at 35, 55 and 75ays after sowing (DAS) and mung bean plants were sampledt 25, 45 and 65 DAS for biochemical analysis and biomassstimation. Final harvest was carried out at complete maturity
.e. 120 DAS for wheat cultivars and 85 DAS for mung beanultivars for estimation of biomass.
For the estimation of chlorophyll and carotenoid contents,.1 g leaf samples (third leaf below the apex of the stem)

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ig. 3. Age wise changes in ascorbic acid content of control and sUV-B exposed Talviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (bars repr


ere placed in 10 ml of cold 80% acetone in a stopperedube overnight in a refrigerator at 4◦ C. It was then homoge-ized and centrifuged at 6000 × g for 15 min. Absorbance ofeaf extract was measured on a UV–vis. spectrophotometerSystronics, model 117, India) at 480 and 510 nm wave-engths for carotenoids and 645 and 663 nm wavelengthsor chlorophyll determination. The amount of total chloro-hyll and carotenoid were calculated by using the formulaeeveloped by Maclachalan and Zalik (1963) and of Duxbury
nd Yentsch (1956), respectively. The methods of Keller andchwager (1977) and Fahey et al. (1978) were used for thextraction and determination of ascorbic acid and total thiolontents, respectively.
riticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv.esent ± 1S.E.).

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Superoxide dismutase (SOD) activity was measured byhe method of Beauchamp and Fridovich (1971). Catalasend peroxidase enzyme activities were determined using theethods of Kar and Mishra (1976) and Britton and Mehley

1955), respectively.For total biomass, randomly sampled plants were oven

ried at 80 ◦C till a constant weight was achieved. Care waslso taken to avoid sampling of plants from the edge of treatedlots and there were no border rows between the sampledlants.

.5. Statistical analysis

Differences between control and UV-B exposed plants
ere determined by DMRT through SPSS software (SPSS
nc., Version 10.0). Multivariate analysis was done to identifyignificant effects and interactions among plant age, sUV-Bnd mineral nutrients.

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ig. 4. Age wise changes in total thiol content of control and UV-B exposed Tritalviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (bars repr


. Results

.1. Photosynthetic pigments

Total chlorophyll contents reduced in wheat and mungean cultivars after exposure to sUV-B irradiation except at 75AS in wheat cultivar grown without fertilizers. Infact they

howed slight increment in the chlorophyll contents. How-ver, nutrient amended and sUV-B exposed plants showed aower percentage of chlorophyll damage than sUV-B exposedlants grown without additional nutrients as compared toheir respective controls except in wheat cultivar HD2329t 75 DAS and both the cultivars of mung bean at 65 DASFig. 1). Maximum chlorophyll contents were noticed at F1
reatment in both the crop plants at all ages except at 65AS in mung bean cultivars. Reductions in chlorophyll con-
ents due to sUV-B were ranged from 10.9 to 31.1% in wheatultivars and from 5.1 to 23.4% in mung bean cultivars. Mul-

icum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv.esent ± 1S.E.).

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ivariate analysis showed significant effect due to plant age,UV-B and fertilizer on chlorophyll content in both cultivarsf wheat and mung bean except Malviya Janpriya for sUV-BTable 1). Carotenoid contents were also affected adverselyy sUV-B in fertilizer amended and non-amended plants ofoth cultivars of wheat and mung bean (Fig. 2). Mineralutrients amended and sUV-B exposed plants showed morearotenoid contents than their respective controls. Analysis ofariance showed significant variations in carotenoids due tolant age, sUV-B and fertilizer dose of both cultivars of wheat
nd mung bean except cultivar Malviya Jyoti for sUV-BTable 1).
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ig. 5. Age wise changes in superoxide dismutase of control and sUV-B exposed Talviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (bars repr


.2. Metabolites

Foliar ascorbic acid contents increased at successiverowth stages of both the crops. Application of mineral nutri-nts increased ascorbic acid contents in all cultivars at alllant ages (Fig. 3). Exposure to sUV-B irradiation resulted inecrease of ascorbic acid contents in both wheat and mungean cultivars at all sampling dates. Percent decreases of

ontents was found in mung bean cultivar Malviya Janpriyat 45 DAS in fertilizer amended (15.1%) and non-amended

riticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv.esent ± 1S.E.).

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lants (27.1%) exposed with sUV-B. Multivariate analysishowed significant variations in ascorbic acid content due tolant age, sUV-B and fertilizer dose. Interactions betweenlant age × sUV-B treatment in Malviya Janpriya, plant age

fertilizer dose in HD 2329 and HUW 234 were also sig-ificant (Table 1).

Total thiol contents of wheat and mung bean plantsncreased with successive growth stages (Fig. 4). Treatmentf sUV-B and application of mineral nutrients also increasedhe thiol content at all ages in all cultivars. Increases in thiolontents due to sUV-B in wheat were 27.0 and 30.4% in HD
329 and 24.5 and 28.1% in HUW 234 at 75 DAS and 27.8nd 36.1 % in Malviya Jyoti and 22.4 and 29.1% in Malviyaanpriya at 65 DAS in fertilizer amended and non-amendedlants.
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ig. 6. Age wise changes in catalase activity of control and sUV-B exposed Triticumyoti and Malviya Janpriya) cultivars with and without nutrients (bars represent ± 1


.3. Enzyme activity

Activity of peroxidase and SOD increased, while catalasectivity decreased in sUV-B exposed cultivars of wheat andung bean plants. Maximum increase in SOD activity due to

UV-B was observed in fertilizer amendment plants (Fig. 5).atalase activity was higher in mineral nutrient amendedlants as compared to the control ones (F0) at all the plantges. Minimum activity of catalase was observed in mungean cultivar Malviya Janpriya at 25 DAS and wheat culti-ar HD2329 at 75 DAS at F0T treatment (0.014 �M H2O2
ecomposed min−1 g−1 fresh leaf) (Fig. 6). Maximum perox-dase activity was found at F1T treatment in all the cultivarsFig. 7). Analysis of variance for catalase and peroxidasectivity showed significant variations in both cultivars of
aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv. MalviyaS.E.).

S.B. Agrawal, D. Rathore / Environmental and Experimental Botany 59 (2007) 21–33 29

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ig. 7. Age wise changes in peroxidase activity of control and sUV-B expoalviya Jyoti and Malviya Janpriya) cultivars with and without nutrients (b

heat and mung bean due to plant age, UV-B treatmentnd fertilizer dose (Table 2). In the present study a clear cutntagonistic interaction was observed between nutrients andUV-B as ample nutrients supply compensate for/or protecthemselves from physiological and biochemical effects ofUV-B.

.4. Biomass

Biomass of both cultivars of wheat and mung beanncreased with successive growth stages. Application of min-ral nutrients also increased biomass of the plants at each
tage of growth (Fig. 8). All plants were negatively influ-nced by the treatment with sUV-B, but inhibition in biomassccumulation was less in nutrients applied plants than theirespective controls (plants grown without recommended dose
4

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ticum aestivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv.esent ± 1S.E.).

f nutrients). Mung bean cultivar Malviya Janpriya showedigher sensitivity to sUV-B, showing maximum reduction iniomass Variations in biomass were significant due to plantge, UV-B treatment, fertility level and interactions betweenlant age ×sUV-B and plant age × fertility levels in bothheat and mung bean cultivars (Table 2). The results clearly

howed that better nutrient availability may reduce the poten-ial damage caused by sUV-B, indicating strong interactionsetween the treatments.

. Discussion

Results of the present investigation showed a negativenfluence of sUV-B radiation on photosynthetic pigments and

30 S.B. Agrawal, D. Rathore / Environmental and Experimental Botany 59 (2007) 21–33

Table 2F-ratios and levels of significance of three way ANOVA test for metabolites and biomass of Triticum aestivum L. (cv. HD 2329 and HUW 234) and Vignaradiata L. (cv Malviya Jyoti and Malviya Janpriya) plants

Cultivars Parameters Factor

Plant age(A)

UV-B treatment(T)

Fertilizer dose (F) A × T A × F T × F A × T × F

HD 2329 Superoxide dismutase 242.54*** 33.73*** 63.16*** 1.98NS 0.48NS 0.12NS 00NS

Catalase activity 243.56*** 41.30*** 305.40*** 00NS 3.86* 4.59* 0.2NS

Peroxidase activity 280.92*** 176.31*** 43.61*** 6.36** 0.02NS 2.22NS 0.14NS

Total biomass 796.33*** 28.79*** 163.63*** 10.61*** 56.88*** 0.06NS 0.06NS

HUW 234 Superoxide dismutase 21.36*** 10.56** 7.23* 0.33NS 0.52NS 0.14NS 0.001NS

Catalase activity 687.62*** 69.62*** 64.62*** 0.93NS 2.69NS 2.59NS 0.13NS

Peroxidase activity 429.56*** 456.58*** 129.71*** 26.69*** 53.82*** 4.25* 3.89*

Total biomass 947.79*** 13.65*** 222.24*** 8.51** 126.12*** 0.03NS 0.02NS

Malviya Jyoti Superoxide dismutase 39.6*** 172.13*** 38.29*** 3.18NS 0.95NS 5.77* 0.46NS


Peroxidase activity 410.73*** 178.94*** 99.1*** 14.54*** 4.09* 7.84** 1.38NS


Malviya Janpriya Superoxide dismutase 8.13** 48.06*** 0.89NS 0.62NS 0.10NS 3.29NS 0.03NS


Peroxidase activity 96.8*** 120.71*** 136.03*** 0.8NS 4.46* 20.82*** 0.34NS


NS: not significant.*

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iomass accumulation of all the test cultivars. The antioxida-ive capacity also altered in experimental plants under UV-Bxposure. Teramura and Sullivan (1994) found reductionn concentration of photosynthetic and accessory pigmentspon UV-B exposure. Reduction in chlorophyll content maye ascribed to the inhibition of its biosynthesis or the degrada-ion of the pigment and their precursors such as protochloro-hyllide, protochlorophyll, etc. (Teramura, 1983; Teramurand Sullivan, 1994). Santos et al. (2004) also found a signifi-ant decrease in total chlorophyll content of potato leaves asompared to unexposed ones. Addition of mineral nutrientsesulted in an increase of chlorophyll content in both UV-Bxposed and unexposed plants, which may be due to avail-bility of higher N for synthesis of pigments. N limitationeduced chlorophyll content (Rousseau and Reid, 1990).

Carotenoids protect chlorophyll from photooxidativeestruction, thus reduction in carotenoids could have seri-us consequences for UV-B radiation effects on chlorophylligments. In the present investigation, significant reductionf carotenoids was observed only at early age, but afterwardseduction was non-significant showing adaptation of culti-ars to sUV-B radiation. Liu et al. (1995) reported increasedarotenoid content in primary leaves of barley after exposureo UV-B. Ambasht and Agrawal (1998), however, reported7% reduction in carotenoid content in Sorghum vulgarelants after 60 days of UV-B exposure. Contrary to this,
ncrement in carotenoid content may represent a biochem-cal response to alleviate UV-B stress because of its role inhe photoprotection of the photosynthetic system by dissipat-ng excess excitation energy through the xanthophylls cycle
awti

Demmig-Adams and Adams, 1992). Higher concentrationf carotenoids was found in both sUV-B exposed and unex-osed plants under elevated mineral nutrients.

Ascorbate is a major primary antioxidant, reacting directlyith hydroxyl radicals, superoxide and singlet oxygen, and is

lso a powerful secondary antioxidant, reducing the oxidizedorm of �-tocopherol. Increase of foliar ascorbic acid concen-ration in sUV-B exposed plants suggested a defense strategyf cultivars to sUV-B stress. The destruction of H2O2 is themportant function of the plant peroxidases that use ascorbiccid as a hydrogen donor. Increment in peroxidase activitylong with ascorbic acid content suggested a higher potentialf H2O2 destruction vis-a-vis greater protection to the plants.

Increase of thiol content in all the cultivars after sUV-Bxposure was observed in the present study. Ascorbate andlutathione are closely related, since both are constituents ofhe antioxidative ascorbate-glutathione cycle, which detoxi-es hydrogen peroxide through a series of enzyme reactions.alatro et al. (2001) reported increased thiol content in soy-ean leaves with increasing UV-B intensity. Massi et al.2002) also reported increased levels of cysteinyl-glycinelow molecular weight thiol) in maize, which is a possi-le degradation product of glutathione, pointing towards anncreased turnover of glutathione due to sUV-B.

Superoxide dismutase, peroxidase and catalase are keynzymes of the antioxidant defense system. SOD acceler-
tes the conversion of superoxide to hydrogen peroxide,hile catalase and peroxidase catalyses H2O2 breakdown. In
he present investigation, activities of SOD and peroxidasencreased, while catalase decreased. Activation of enzymes

S.B. Agrawal, D. Rathore / Environmental and Experimental Botany 59 (2007) 21–33 31

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ig. 8. Age wise changes in total biomass of control and sUV-B exposed Tryoti and Malviya Janpriya) cultivars with and without nutrients (bars repre

as more in wheat as compared to mung bean cultivars, sug-esting a strong antioxidative scavenging capability of theormer. Costa et al. (2002), however found reduction in SODctivity leading to O2

•− accumulation, which therefore coulde responsible for decrease of chlorophyll in sUV-B exposedunflower cotyledons. Inhibition of catalase activity may alsoe ascribed to enzyme consumption to detoxify H2O2 ornzyme inactivation (Ambasht and Agrawal, 2003). Simi-ar findings were also reported by Agrawal (2002) in Vicia
aba. Alexieva et al. (2001) reported increments of catalasend peroxidase activities in pea plants opon UV-B exposurehile increase of catalase and decrease of peroxidase activityas observed for wheat plants.
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estivum L. (cv. HD 2329 and HUW 234) and Vigna radiata L (cv. MalviyaS.E.).

Though, the antioxidant defense system was activated inll the cultivars, reductions in biomass were observed in allultivars due to sUV-B exposure. Change in biomass accu-ulation is an important measure to assess sUV-B sensitivity,

ince this parameter reflects the cumulative effect of manymall disruptions in plant function. Reduction in biomass dueo sUV-B was of lower magnitude in plants supplementedith mineral nutrients. Musil and Wand (1994) found simi-

ar results for the winter ephemeral Dimorphotheca pluvialis
uring the early developmental stage upon UV-B exposure.owever, interaction between nutrient availability and UV-Bas not present in mature leaves. The present investigation
howed more damaging effect of sUV-B on biomass of mung

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ean cultivar Malviya Janpriya suggesting its higher sensitiv-ty to sUV-B. However, this cultivar did not show the highestecline in chlorophyll content. Ghisi et al. (2002) also foundeduced fresh and dry weights of UV-B exposed barley plants,ut the reduction was not correlated with decline in photosyn-hesis rate. Quaggiotti et al. (2004) observed no difference iniomass, while Ambasht and Agrawal (1995) found higheriomass in UV-B treated Zea mays as compared to theirespective controls. Teramura et al. (1991) showed signifi-ant reduction in total biomass in 6 out of 16 rice cultivarscreened for UV-B sensitivity.

The activation of the free radical scavenging system due toUV-B highlighted the differential role of each component inhe UV-B stress acclimation process in wheat and mung beanultivars. This coordinated defense mechanism helped thelants to recover in terms of growth after stress cycle. sUV-Btress showed an increase in tested enzymes along with totalhiols and ascorbic acid. Further, these antioxidants increasedith application of nutrients, increasing the resistance of both

ultivars. Lavola et al. (2003) revealed that high nutrient avail-bility increases the resources in seedlings and thereby, therowth and the investment for protection would be greaternder the high nutrient than moderate nutrient level.

. Conclusion

Wheat cultivars HD 2329 and HUW 234 and mung beanultivars Malviya Jyoti and Malviya Janpriya are sensitiveo sUV-B. Inter- and intra-specific variations were presentn tested cultivars in response to sUV-B radiation and it wasell correlated with free radical scavenging capacity of tested

ultivars. However, adaptations or acclimation to photoox-dative stress is multifactorial and many factors are involvedn the overall defense strategy of the plants. Applicationf recommended dose of mineral nutrients showed signifi-ant positive response by ameliorating the negative effects ofUV-B radiation by increasing the levels of antioxidants. Tonderstand the exact mechanism of tolerance more detailednvestigations are required under field conditions on growth,hysiological and biochemical responses and genetic controlo sUV-B radiation.

cknowledgements

The authors are thankful to FFC section, Indian Councilf Agricultural Research (New Delhi) for financial assistancen the form of a research project and to authorities of Alla-abad Agricultural Institute, Deemed University, Allahabad,or necessary experimental facilities.

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