Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B

    Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B Geeta Yadav, Prabhat Kumar Srivastava, Parul Parihar, Sanjesh Tiwari, Sheo Mohan Prasad PII: DOI: Reference:

S1011-1344(16)30493-6 doi:10.1016/j.jphotobiol.2016.10.011 JPB 10610

To appear in: Received date: Revised date: Accepted date:

27 June 2016 7 October 2016 11 October 2016

Please cite this article as: Geeta Yadav, Prabhat Kumar Srivastava, Parul Parihar, Sanjesh Tiwari, Sheo Mohan Prasad, Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B, (2016), doi:10.1016/j.jphotobiol.2016.10.011

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ACCEPTED MANUSCRIPT Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B

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Authors: Geeta Yadav1, Prabhat Kumar Srivastava1,2, Parul Parihar1, Sanjesh Tiwari1, Sheo

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Mohan Prasad1*

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Affiliations:

1. Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany,

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University of Allahabad (A Central University of India), Allahabad-211 002, India 2. Pt. Ravi Shankar Tripathi Government Degree College, Bhaiyathan, Surajpur-497

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231, C.G., India E-mails:

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[email protected] (PKS) Mob: +91-9415241842

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Abstract

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*Corresponding author

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*[email protected] (SMP) Mob: +91-9450609911

In order to know the impact of elevated level of UV-B on arsenic stressed Helianthus annuus L. var. DRSF-113 plants, certain physiological (growth‒ root and shoot lengths, their fresh masses and leaf area; photosynthetic competence and respiration) and biochemical parameters (pigments‒ Chl a and b, Car, anthocyanin and flavonoids; reactive oxygen species‒ superoxide radicals, H2O2; reactive carbonyl group, electrolyte leakage; antioxidants‒ superoxide dismutase, peroxidise, catalase, glutathione-S-transferase, proline) of their seedlings were analysed under the simultaneous exposures of two arsenic doses (6 mg kg-1 soil, As1; and 12 mg kg-1 soil, As2) and two UV-B doses (1.2 kJ m-2d-1, UV-B1; and 3.6 kJ m-2d-1, UV-B2). As1 and As2 alone declined all the studied growth parameters‒ along with

ACCEPTED MANUSCRIPT photosynthetic pigments which were further aggravated after the simultaneous exposures of predefined levels of UV-B. Each As exposure was accompanied by significant accumulation

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of As in root, shoot and leaves and was substantiated by simultaneous exposures of UV-B

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doses which manifested into suppressed growth, decreased chlorophyll contents and

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photosynthesis. In similar conditions, other photo-shielding pigments, viz. carotenoids, anthocyanin and flavonoids along with respiration and oxidative stress markers such as O2•¯, H2O2; and indicators of cell membrane damage like MDA (malondialdehyde), RCG (reactive

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carbonyl group), electrolyte leakage were enhanced by As, and became more pronounced

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after the simultaneous exposures of UV-B doses. As doses stimulated the activities of SOD, POD, CAT, GST and Pro which got further accelerated after the simultaneous exposures of

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UV-B doses.

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Keywords: Antioxidants; Arsenic; Helianthus annuus; Oxidative damage; Oxygen toxicity

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UV-B. 1. Introduction

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Arsenic contamination to farmlands has become a global problem in recent times. Geochemical weathering of rocks, volcanism and microbial activities are some of the natural processes via which its mobilization occurs into the environment (see review by Singh et al. [1]). Food chain is infiltrated by As through As loaded groundwater and industrial and municipal waste contaminated water used for irrigation purposes. As also penetrates the food chain through the usage of fertilizers and herbicides (arsenicals) in agricultural fields. Higher levels of As than permissible level (20 mg kg-1) have been reported in several countries extending from Bangladesh, India, South-East Asian countries, China and Japan to Canada, USA, Mexico and Argentina, encompassing a total of 21 countries [2]. The worst situation is

ACCEPTED MANUSCRIPT prevailing in Bangladesh followed by West Bengal province of India where the concentration of As in drinking water has been reported as much as 50 μg L-1[3].

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As severely intoxicates plants via various physiological and biochemical anomalies

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and reduces their growth and development. Toxicity symptoms range from biomass reduction

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to morphological impairments leading to the loss in fruit and grain yield which culminates into the complete death of the plants [4, 5]. Severe toxic effects of As change the

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concentration, accumulation and translocation of nutrient elements in plants, inhibits seed germination and increases As levels in the edible parts of vegetables [5].

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As exists in two inorganic forms, arsenite (AsO33−) and arsenate (AsO43−) (referred as AsIII and AsV, respectively). AsIII and AsV are inter-convertible to each other and are readily

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taken up by the plant cells through phosphate transporters and quaglyceroporins, respectively

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[6]. Generally, AsIII is considered to be the most phytotoxic form while AsV is the

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predominant phyto-available form in aerobic conditions. Proteomic analyses revealed that larger number of genes and proteins were differentially expressed following exposure to AsV

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when compared to AsIII [7].

There are substantial pieces of evidence that exposure of plants to inorganic As does result in the over-generation of reactive oxygen species (ROS, e.g. singlet oxygen, 1O2; superoxide radical, SOR, O2•¯; and hydrogen peroxide, H2O2) resulting into the oxidative stress [8]. ROS may also be generated during As detoxification process when AsV reduces into AsIII [4]. Higher concentrations of ROS than the threshold limit disrupt pigments, proteins, lipids and nucleic acids thus affect the normal cellular metabolism, a condition known as oxygen toxicity. The balance between ROS production and their scavenging determines the successful survival of an organism. Under stress conditions, for example, under As toxicity, organisms have to activate the antioxidative defence system orchestrated

ACCEPTED MANUSCRIPT by number of enzymes like superoxide dismutases (SOD, EC 1.15.1.1) peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6) and glutathione-S-transferase (EC 2.5.1.18) having

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capability to detoxify ROS.

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In natural conditions, generally, a multitude of environmental stressors occur

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concomitantly. Sunlight is indispensable for photosynthetic organisms and therefore they are inevitably exposed to UV-B radiation. UV-B is that subpart of ultraviolet electromagnetic

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spectrum which falls in the range of 280-315 nm wavelength. Sufficient amount of UV-B radiation comes on the earth surface due to the depletion of stratospheric ozone layer. UV-B

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that reaches the earth surface has the highest energy among all the incident wavelengths of sunlight spectrum because it has the least wavelength (see reviews by Kataria et al. [9],

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Parihar et al. [10, 11]. The ambient UV-B has significant inhibitory effects on plants’

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development and morphology [12, 13]. Prevailing UV-B can alter the physiology either

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through direct damage or via various regulatory effects [14]. Since, UV-B also over-generates ROS, so can potentially damage essential biomolecules including genetic materials, lipids and proteins thereby disintegrating membranes [15].

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Since sunflower produces edible oil and some of the major sunflower producing areas in India encompasses one of the worst As polluted soils of the world. So, sunflower was taken as the experimental material of the present investigation. Simultaneous incidence of UV-B and As treatment to Helianthus may augment the negative and inhibitory effects on morphology and physiology of Helianthus leading to more adverse impact on productivity and biomass production. Therefore, this study was aimed to investigate the interactive effects of As and UV-B in sunflower seedlings under ambient light on certain growth, morphological, physiological and biochemical parameters.

ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Plant material and growth conditions:

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Sunflower seeds (Helianthus annuus L. var. DRSF-113) were obtained from Directorate of

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Oilseed Research, Rajendranagar, Hyderabad, 500030, Andhra Pradesh, India. Seeds were

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surface sterilized, allowed to germinate and then transferred to plastic pots having equal amount of acid-washed sterilized sand. For details of growth conditions our previous published paper Yadav et al. [16] can be consulted. The seedlings were supplemented with

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half-strength Hoagland’s nutrient medium after the emergence of first leaves. Sodium

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arsenate (Na2HAsO4.7H2O) containing AsV was used as a source of As. After 13th day of growth seedlings were treated with 6 and 12 mg As kg-1 soil (abbreviated as As1 and As2,

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environmentally relevant dose). Two levels of enhanced UV-B radiation, low (UV-B1:

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ambient +1.2 kJ m-2 day-1) and high (UV-B2: ambient + 3.6 kJ m-2 day-1) was provided by UV-B lamps (Q-Panel Co, UV-B-313 flourescent lamps, OH, USA) from 09:30 hrs (3.5 h

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after the beginning of the photoperiod) to 15:30 hrs. Each sample was simultaneously receiving ambient level of UV-B at Allahabad, India (8.2 kJ m-2day-1).

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2.2. Measurement of growth and related parameters: Root Fresh Mass (RFM), Shoot Fresh Mass (SFM), Root Length (RL), Shoot Length (SL) and Leaf Area: Fresh mass of the roots and shoots were weighed using digital balance (Contech-CA 223, India) after 48 h of treatment. Shoot length and root length,of the seedlings were measured by using meter scale. The leaf area of treated and untreated seedlings was measured using Leaf Area Meter (Model 211, Systronics, India). 2.3. Estimation of As content

ACCEPTED MANUSCRIPT The content of As in root, shoot and leaf samples was determined after due digestion process in nitric acid-perchloric acid mixture (5:1, v/v) by using inductively coupled argon plasma-

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atomic emission spectroscopy (ICAP-AES).

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2.4. Estimation of pigments: chlorophyll a, b, carotenoids, anthocyanin and flavonoids

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The amount of chlorophylls (Chl a and b) and carotenoids were calculated by using the equations of Lichtenthaler [17] after extracting definite amount of leaves from treated and

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untreated seedlings in 80% acetone. UV-Visible double beam spectrophotometer (UV-Vis 1700, Shimadzu, Japan) was used to read optical densities.

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Anthocyanin was estimated in fully exposed leaves of treated and untreated seedlings by the method of Wanger [18].

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For flavonoids content, leaf discs were kept in acidified methanol (CH3OH: H2O: HCl

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:: 78: 20: 2; v/v) for 24h at 4ºC and the absorbance of filtered extract was measured at 320

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nm. Flavonoids contents are expressed as absorbance g-1 fresh mass of tissue at 320 nm. 2.5. Measurement of net photosynthetic rate, respiration photosynthetic electron transport activities and restoration of PS II by exogenous electron donors:

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Net photosynthetic oxygen yield was measured in leaf discs of treated and untreated samples by the method of Kurra-Hotta et al. [19] by using Clark type oxygen electrode (Digital Oxygen System, Model-10, Rank Brothers, UK) and was expressed as mmol O2 evolved m-2 h-1.Respiration was also measured with the similar method but expressed in the terms of mmol O2 consumed m-2 h-1. Polarographic assay was done for photosynthetic electron transport activities (PS II, PS I and whole chain) in the leaves of various samples. PS II was assayed in the terms of O2 evolution. PS I and whole chain activities were assayed in the terms of O2-consumption. Spectrophotometric assay of PS II activity as DCPIP photoreduction was monitored by

ACCEPTED MANUSCRIPT measuring changes in the absorption at 600 nm in the absence and presence of various exogenous electron donors i.e. MnCl2, diphenyl carbazide (DPC) and NH2OH separately.

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2.6. Estimation of SOR and H2O2, RCG, MDA and EL

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Accumulation of SOR was measured by the method of Elstner and Heupel [20] with some

standard curve prepared by using sodium nitrite.

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modifications as described by Jiang and Zhang [21]. The SOR content was quantified from a

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Hydrogen peroxide contents from control and treated seedlings were estimated by the method of Velikova et al. [22]. The concentration of H2O2was calculated by using a standard

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curve prepared with H2O2.

Oxidative damage to proteins was estimated in the terms of reactive carbonyl groups

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using di-nitrophenylhydrazine according to the method of Levine et al. [23] with some

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modification. The amount of reactive carbonyl groups formed was calculated using molar

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absorption coefficient of 22,000 M-1 cm-1. Lipid peroxidation was determined in the terms of MDA (malondialdehyde) content, a

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product of unsaturated fatty acid peroxidation known as 2-thiobarbituric acid (TBA) reactive metabolite [24]. The MDA concentration was calculated using the extinction coefficient 155 mM1 cm-1.

The intactness of plasma membrane in plants of each set was estimated in terms of EL by the method of Gong et al. [25] with the digital Conductivity Meter (Century CC-607, India). The percentage of electrolyte leakage (EC) was calculated as follows:

EC=

EC1 100  %  . EC2

2.7. Assay of antioxidants: Enzymatic antioxidants: SOD, POD, CAT and GST

ACCEPTED MANUSCRIPT Superoxide dismutase activity was estimated by measuring decrease in the reduction of nitroblue tetrazolium chloride (NBT) by the method of Giannopolitis and Ries [26]. One unit

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of SOD is defined as the amount of enzyme which decreases the NBT reduction by 50%

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under the defined conditions.

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Peroxidase activity in the leaves of each set of seedlings was determined according to Zhang [27] using extinction coefficient 25.5 mM-1 cm-1. Enzyme activity was calculated in

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the terms of Unit g-1 FM. One unit of POD activity is the amount of enzyme oxidizing 1 nM guaiacol min-1.

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CAT activity was determined in the terms of decrease in absorbance due to decomposition of H2O2which was recorded at 240 nm using the extinction coefficient of 39.4

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mM-1cm-1 (Aebi, 1984) [28]. One unit of CAT activity is the amount of enzyme dissociating 1

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nmol H2O2 min-1.

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Glutathione-S-transferase activity was measured by the method of Habig et al. [29] using CDNB (1-chloro-2,4-dinitro benzene) as a substrate. The activity of GST was

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calculated using the extinction coefficient 9.6 mM-1 cm-1. One unit of GST activity is expressed as 1 nmol CDNB conjugates min-1. Non-enzymatic antioxidant: Pro Proline content in leaf homogenates was estimated by the method of Bates et al. [30] with the help of standard curve. 3.

Statistical analysis

The results presented are the means of six independent experiments. In each experiment, ten seedlings were randomly selected for the determination of growth parameters while three seedlings were used for other parameters. One-way ANOVA was performed to test

ACCEPTED MANUSCRIPT significance level (Duncan’s multiple range test, DMRT) at p<0.05. Two way ANOVA test was also performed to show the differential action of As and UV-B alone as well as in

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Results:

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4.

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combination. SPSS-16 software was used for DMRT.

4.1. Growth behaviour

Various parameters pertinent to growth performance, viz. root fresh mass (RFM), shoot fresh

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mass (SFM), root length (RL), shoot length (SL), leaf area (LA) were recorded under the twin

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stressors of As and UV-B and have been shown in Fig. 1. Both the As doses and UV-B2 dose significantly declined RFM, SFM and LFM and the degree of damage was in dose

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proportional manner, while UV-B1 did not cause any significant decline in these parameters.

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Further, RFM and SFM were synergistically affected under the simultaneous influence of the twin stressors (except As1+UV-B2 on RFM). Contrarily, RL was significantly affected by

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either of the As doses while any of the UV-B doses couldn’t cause any significant inhibition in RL. Low dose of As or UV-B couldn’t decline shoot length while the similar dose had a

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substantial impact, about 9% and 35% reduction in SL and LA, respectively. The extent of damage on RL and SL became synergistic after the simultaneous exposures of both the stressors, while the impact of simultaneous exposure on LA did not have synergistic effect. Fig. 1 clearly epitomizes that both the doses of As affected RFM and RL more vigorously than they had affected SFM and SL. Contrarily, both the doses of UV-B more affected SFM and SL than they affected RFM and RL. But above all, it was the LA which was the most severely affected by either of the AS or UV-B doses. Whatever be the pattern, in most of the cases the damage was exaggerated approaching synergistic effects. 4.2. As content

ACCEPTED MANUSCRIPT As the amount of As was increased, the accumulation of As increased in the plant tissues and the accumulation of As followed the pattern– root>shoot>leaf. Further, Fig. 2 says that

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whenever seedlings were exposed to UV-B simultaneously with As doses, the seedlings

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became more prone for the As accumulation and subsequently accumulated more As.

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4.3. Pigments: chlorophyll a, b, carotenoids, anthocyanin and flavonoids Results pertaining to Chl a, b, carotenoids, anthocyanin and flavonoids contents have been

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depicted in Fig. 3. The levels of Chl a and b showed decreasing trend and the reduction was linear with increasing doses of As and UV-B. As2 and UV-B2 doses significantly decreased

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the Chl a and b contents while UV-B1 did not pose any significant alteration in Chl a or b. Though, when the seedlings were exposed to As together with UV-B doses, the decrease in

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seldom synergistic with Chl b.

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the amount of chlorophyll pigments was synergistic in the case of Chl a while they were

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Carotenoids, being the anti-stress pigment exhibited increment after single treatment of any of the stressors; however, it was significantly damaged after As2+UV-B1, As1+UV-B2

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and As2+UV-B2 treatments. As1 and As2 alone caused 6 and 13% increase in anthocyanin level while UV-B1 and UV-B2 alone caused 21 and 26% increase in anthocyanin level. When both the stressors applied in combinations, the level of anthocyanin increased with the synergistic effects. As1 and As2 alone caused 9 and 16% increase in flavonoids contents over the value of control while UV-B1 and UV-B2 alone caused an enhancement of 17 and 28%. Flavonoids contents increased synergistically when both the stressors were applied in combinations. 4.4. Net photosynthetic rate, respiration, photosynthetic electron transport activities and restoration of PS II

ACCEPTED MANUSCRIPT Results pertaining to the net photosynthetic oxygen yield and respiration have been portrayed in Fig. 4. The net photosynthetic oxygen yield considerably declined in Helianthus annuus

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seedlings when treated with either doses of As or UV-B. As1 and As2 alone caused an

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inhibition of about 5 and 10%, respectively. UV-B1 and UV-B2 also caused an inhibition of

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about 5 and 12%, respectively. When the two stressors were given in combination, the net photosynthetic oxygen yield declined with the synergistic effects, ranging from 16 to 40%.

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The rate of respiration was increased with the single as well as simultaneous exposures of As and UV-B doses. As1 caused 9% increment and As2 caused 14% increment

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while both the doses of UV-B could elevate respiration level by about 10 and 12%, respectively. When the test seedlings were exposed to As and UV-B jointly, the rate of the

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respiration did increase more than either of the individual values but never appeared to

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approach the synergistic levels, despite decreased for As2+UV-B2 (Fig. 4).

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Results pertaining to photosynthetic electron transport activities in PS II, PS I and whole chain system have been shown in Table 1. These activities were inhibited by both the stressors, alone as well as in combination. These activities showed maximum reductions at

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As2+UV-B2 dose while As1+UV-B1 posed the least effect among various combined treatments. The inhibition level followed the sequence: PS I
ACCEPTED MANUSCRIPT treated with low dose of As1 however, the least restoration was observed under the highest stress condition (As2+UV-B2).

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4.5. Reactive oxygen species and indices of oxidative damage: (SOR, H2O2, MDA, RCG and

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EL)

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Results pertaining to production of O2•¯, H2O2, MDA, RCG and electrolyte leakage have been depicted in Fig. 5. Both the doses of As and UV-B produced increase in O2•¯ and H2O2,

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MDA, RCG production and the production of ROS became synergistic in most of the

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combinations. Similar trend of results was found with EL. 4.6. Enzymatic antioxidants (SOD, POD, CAT and GST)

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Results presented in the Fig. 6 represent SOD, POD, CAT and GST activities. After the

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exposure of As and UV-B alone and in combination, SOD, POD, CAT and GST activities

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substantially. When both the stressors were applied in combinations, in most of the cases the activity of enzymatic antioxidants raised with the synergistic effects.

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4.7. Non-enzymatic antioxidant: Pro Similarly, as in the case of enzymatic antioxidants, proline content also substantially increased with As and UV-B exposures alone and in combination but when both the stressors applied in combination, they additionally increased the Pro content, but not with the synergistic effects. Table 3 shows two-way ANOVA test of the twin stressors and the studied parameters in H. annuus seedlings. The data revealed that both the stressors i.e. As and UV-B significantly affected all the studied parameters but the effect was more intensified when the stressors were combined. Regarding the combined effect of twin stressors, there was significant interaction for photosynthesis, root length, leaf area and accumulation of As in root and shoot and also for the oxidative stress parameters and their indices. This significant

ACCEPTED MANUSCRIPT interactive effect of twin stressors suggests their synergistic effect thereby damaging the tested plants more intensively. The antioxidant system i.e. CAT, GST and proline was also

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significantly affected under combined exposures of both the stressors that indicate towards

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the inefficiency of these antioxidants to remove the ROS and their by-products, thereby

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causing greater damage under the twin stressors. 5. Discussion

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Increased As levels have been found to affect the normal growth and development process of the plants and culminate into reduction in the yield of many plants [31]. The root-shoot

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elongation along with biomass production was significantly decreased in different plant species owing to the As-induced morphological and physiological disorders [8, 32]. The

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effect of As was more prominent on the root length compared to the shoot length because the

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root system was in direct contact with As. Similarly, the impact of UV-B was more

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prominent on the shoot system since the shoot system was directly exposed to UV-B light (Fig. 1). Further, reductions in root growth directly impacts shoot growth and vice-versa. Earlier reports in other plant species viz. Luffa acutangula and Vigna mungo also showed

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similar inhibitory effects of As on root and shoot lengths [33, 34]. Analogous nature of As and phosphate [4] leads to reduce phosphate uptake and uncouples oxidative phosphorylation and restrains plant growth. AsV and AsIII are the main

species of As that are translocated from roots to shoot tissues via the xylem [35]. After the uptake of AsV by root cells endogenous arsenate reductases reduce AsV to its more toxic form i.e. AsIII [36]. AsIII has high affinity with −sulfhydryl groups of enzymes and tissue proteins and thereby inhibits many key metabolic processes in the cell [6]. The accumulation of As in different plant tissues, root, shoot and leaves may result into considerable reduction in the yield of plants.

ACCEPTED MANUSCRIPT Reduction in biomass accumulation due to UV-B exposure has earlier been found in several plant species which ultimately reduced the crop yield [11, 14]. UV-B stress has also

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been observed to reduce root biomass in other plants [37]. Several studies have shown that

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enhanced UV-B radiation significantly affects plant height and leaf length/area [38]. Direct

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effects of UV-B on shoot system must involve indirect effects on root system. It has also been assumed to be the consequence of UV-B induced changes in hormone metabolism and thereby cell wall loosening [39].

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The deleterious effects on photosynthetic pigments of Helianthus annuus was dose

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dependent. There might be reductions in chlorophyll precursors’ levels under As exposure [21]. Our results are in agreement with some other workers where As has been reported to

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reduce chlorophyll biosynthesis [40]. UV-B also causes reduction in chlorophylls which may

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result in lower level of biomass accumulation [41]. Similar reductions in Chl a and b were observed in Vigna mungo, Vigna unguiculata and Crotalaria juncea after enhanced UV-B

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exposure [34, 42]. UV-B radiation affects the photosynthetic pigments, either through inhibition of their synthesis or effects on the enzymes involved in the chlorophyll

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biosynthetic pathway, ultimately leading to lesser efficiency to photosynthesize. Besides this, UV-B might have destroyed the photostability of chlorophylls and caused down-regulation of genes for Chl a/ b binding proteins, thereby inhibited chlorophyll biosynthesis [43]. The effect of As and UV-B was more pronounced on Chl a followed by Chl b (Fig. 3) because of the selective damage to Chl a biosynthetic process or degradation of its 7 precursors by UVB [44]. In comparison to chlorophyll, anti-stress pigments viz. carotenoids, anthocyanin and flavonoids exhibited increment. Since, Car are anti-stress pigments, shield chlorophyll from the miseries of light, excess of light or UV-B like harmful radiations and also act as antioxidants therefore, it increased up to a certain level in response to a certain amount of

ACCEPTED MANUSCRIPT stress (UV-B1 + As1) in order to protect the chlorophyll molecules but as the amount of stress surpasses the tolerance capacity, carotenoids themselves were damaged as observed in the

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cases of As2+UV-B1, As1+UV-B2 and As2+UV-B2 treatments. Therefore, we obtained

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distinct curves which are far apart from one another (Fig. 3). Enhancement in Car contents

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suggests the As-tolerance strategy of Helianthus which might be an adaptive mechanism to cope against the photo-damage to chlorophyll and photosynthetic apparatus (Fig. 3). Accumulation of flavonoids in the epidermis provides a shield to protect plant from UV-B

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radiation [45].

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Photosynthetic oxygen yield was adversely affected by all the doses of As and enhanced levels of UV-B radiations (Fig. 4). Inhibition in photosynthetic process is the direct

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consequence of alterations in the chlorophyll biosynthesis and loss to the chloroplast ultra-

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structure thus affects biomass accumulation [30]. Inhibition in photosynthetic activities due to As may be correlated with the accumulation of As in leaves. Arsenic may substitute the

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central atom (Mg) of the chlorophyll molecule reducing the photosynthetic rate. The downregulation of chloroplast 29 kDa ribonucleoproteins and alteration in structure and amount of

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large subunit of RuBisCO affects O2 evolution [7]. UV-B-induced inactivation of RuBisCo could be due to modification of the peptide chain, degradation of the protein, and/or diminished transcription of the genes impacting the catalytic capacity of RuBisCo [46]. UVB has also been reported to decline the activity of large sub-unit of RuBisCo in mature leaf of oil seed rape [47]. Despite this, UV-B induced ROS production damages RuBisCo [48]. Decrease in O2 evolution has also been observed in other plants after UV-B exposure [49]. The negative impact of UV-B on chloroplasts ranges from the reduction in their number and size, breakage and swelling of thylakoid membrane due to oxidative damage to lipids and proteins. UV-B exposure may lead to oxidation of photochemical apparatus, primary photochemistry and electron transport; phosphorus deficiency and reduced Mn

ACCEPTED MANUSCRIPT transport [50]. UV-B radiation damages the photosynthetic apparatus of plants at multiple sites.

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UV-B radiation affects photosystem II redox components at both the donor and

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acceptor side, of this the donor side is the most sensitive to UV-B. There are inferences of

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specific destruction of the reduced quinone by UV-B or structural changes in QB binding site (see review by Bornman [51]). The catalytic site contains a manganese cluster that has been proven to split water molecule into O2, protons and electrons. Catalytic site of the water

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oxidation is the most sensitive part of PS II to UV-irradiation and is blocked by it [52].

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Polypeptides D1 and D2 form the apoprotein of the reaction centre complex of PS II. Therefore, structural modification of the D1 and D2 complex could be accompanied by a

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modification of the donor side. Low-temperature EPR spectroscopy showed that the rate of

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the D1 protein loss is slower than the inhibition of oxygen evolution [53]. UV-B irradiation also affects Cyt b-559. Modification and/or inactivation of tyrosine-D, Cyt b-559, and the QA

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Fe2+ acceptor complex are subsequent events that coincide more closely with the UV-Binduced damage to the protein structure of the PS II reaction center [53]. The radiation

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inactivates light harvesting complex II (LHC II) and alters gene expression for the synthesis of PS II reaction centre proteins. This ultimately reduces the photophosphorylation process of light reaction [8]. Likewise in our study, damage to PS II has been found greater than PS I in earlier studies as well [54]. The previous studies suggested that the PS I is the least affected component by UV-B [26]. Artificial electron donors are extremely useful in measuring the electron transport activity of various segments of the photosynthetic electron transport pathway. They are uncharged, redox sensitive compounds, so can easily be transported across the membranes. They donate electrons to different specific sites on the water oxidation side of PS II. Table 2

ACCEPTED MANUSCRIPT clearly epitomizes that NH2OH followed by DPC were more supportive to the PS II system followed by DPC, while MnCl2 could not restore the PS II activity efficiently.

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In contrast to photosynthesis, respiration rate showed significant enhancement in

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Helianthus annuus seedlings up to a certain amount of stress (As1 + UV-B2). Partial

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uncoupling of the electron transport chain takes place following thylakoid membrane disruption under mild stresses. Therefore, increase in O2 uptake was recorded to meet the situation of disrupted respiratory apparatus. But, drop in respiration rate was also observed as

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the amount of stress increased beyond the tolerance limit. Therefore, we get entirely different

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respiratory curves (Fig. 4). Increase in respiration rate might have been an adjustment to meet the demand of ATP for carrying out the basic life processes. Enhanced uptake of O 2 may also

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occur due to excessive formation of ROS under stress conditions. Ahsan et al. [7] while

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working on protein profiling of rice leaves under As stress found that expression of at least 14 proteins, mostly associated with energy metabolism pathways, was either up-regulated or

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down-regulated suggesting that higher energy is required for the activation of the metabolic processes in the leaves exposed to As . AsV acting as a Pi analogue is transported across the

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plasma membrane to cytoplasm via a Pi co-transport system and replaces Pi in ATP to form unstable ADP-As complexes. This leads to the disruption of energy flows in cells [55]. Another notorious effect induced by heavy metal and UV-B is excessive generation of ROS, e.g. O2•¯, •OH, H2O2 [20]. AsV has earlier been reported to induce ROS [8, 33, 41]. ROS is generated probably through the conversion of AsV to AsIII in the cells [4]. Additionally, UV-B might have obstructed the normal reduction path of NADP+ to NADP.2H during photosynthetic electron transport chain and over-generated ROS in As-stressed H. annuus seedlings (Fig. 5). Malondialdehyde (MDA) is a by-product of lipid peroxidation, an indicator of As and UV-B induced oxidative damage in Helianthus annuus seedlings (Fig. 5). Damage to lipids further results in the generation of ROS and subsequent oxidative stress.

ACCEPTED MANUSCRIPT Earlier reports also showed similar results in rice seedlings [8]. Enhanced level of H2O2 coupled with high level of lipid peroxidation might have damaged chloroplasts which led to

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decreased plant biomass and inhibited chlorophyll biosynthesis as well as chlorophyll loss

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[21].

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Proteins that have sulfur-containing amino acids and thiol groups are prone to be attacked by ROS. Protein oxidation inhibits enzymatic activities and increases the susceptibility of membranes towards proteolytic attack. The increased RCG content after As

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and UV-B exposures as observed in present study (Fig. 5) might be due to oxidation of a

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number of amino acids, particularly Arg, His, Lys, Pro, Thr and Trp. UV-B has been reported to increase RCG in the leaves of Vigna unguiculata and Crotalaria juncea [42]. Damage to

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leakage as observed in our study.

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lipids and proteins by ROS may alter cell membrane permeability which leads to electrolyte

Enhanced formation of ROS under stress conditions induces cellular damage and

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protective responses both. Plant cells contain a wide range of antioxidant enzymes such as SOD, POD, CAT, GST etc. and non-enzymatic metabolites, e.g. proline which serve as ROS

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quenchers thus protect cells from oxidative damage [20]. Both the stressors (As and UV-B) either alone or in combination produced stimulating effects on antioxidative enzymes, SOD, POD, CAT and GST and non-enzymatic antioxidant, proline which were directly proportional to ROS formation (Fig. 6). SOD, CAT and POD cooperatively orchestrate against ROS. The SOD activity has also been reported to increase in wheat, rice and maize seedlings after AsIII treatments [32, 33]. SOD dismutates O2•¯, one O2•¯ being reduced to H2O2 and another oxidizes to O2 [56]. This decreases the risk of •OH formation with metal catalyzed Haber-Weiss type reaction. Excess H2O2, a lesser active oxidant, is later scavenged by CAT and POD. CAT is considered one of the best known H2O2 splitting enzymes whereas POD plays an important role in the degradation of lipid peroxides [8]. Catalase is known for

ACCEPTED MANUSCRIPT its unequivocal role in extenuating H2O2-mediated oxidative damage and therefore its formation is up-regulated up to a certain limit of stress. However, the activity of catalase

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diminishes beyond this limit. Therefore, here too, three distinguished curves were obtained

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(Fig. 6). Besides this, enhanced amounts of H2O2 and hence MDA, RCG and electrolyte

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contents symbolize that CAT became inadequate in mitigating H2O2 and preventing membrane damage. In one of the earlier studies decline in CAT activity was observed along with an increase in As concentration in rice [32].

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Products formed by the damage of biomolecules and cell membrane may further

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initiate free radical-based chain reactions. GST participates in the detoxification reaction of these products. Enhancement in GST activity has also been found after Cd application in

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Pisum sativum [57]. In plants exposed to heavy metals, GST may participate in transport of

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phytochelatin-metal complexes to vacuoles [58]. Proline, an amino acid, scavenges free radicals and is supposed to be the effective

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scavenger of •OH for which no enzymatic antioxidants are known to have evolved [32]. Besides, acting as a cytoplasmic osmoticum, proline protects protein against denaturation, and

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behaves as a metal chelator, an antioxidative defence molecule as well as a signalling molecule. The amount of proline has been reported to be raised significantly in wheat seedlings after As III exposure [33]. Exposure of UV-B also enhanced proline content in earlier observations [49]. 5. Conclusion Present investigation is a demonstration of synergistic inhibitory behaviour of enhanced UVB (along with ambient UV-B) under As stressed condition in Helianthus annuus L. seedlings. Different ROS and antioxidants orchestrated in such a way that the impacts of individual stresses were far lesser on various growth and physiological parameters. However, interactive and synergistic impacts were observed on growth and physiological parameters of the

ACCEPTED MANUSCRIPT seedlings when the stressors were combined with each other and hence overall growth and development of Helianthus annuus L. seedlings was impacted far worse than their individual

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effects. If these studies are further combined with transcriptomics, proteomics, metabolomics

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etc. popularly called as ‘OMIC tools’ will add new dimensions in the mechanism of

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interaction of two or more stressors. Acknowledgements:

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The authors are thankful to The Head, Department of Botany, University of Allahabad, Allahabad for providing necessary laboratory facilities. The authors are also thankful to The

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University Grants Commission (UGC), New Delhi and The Council for Scientific and Industrial Research (CSIR) New Delhi for providing financial support for purchasing

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necessary instruments and chemicals, and for financial assistance to Parul Parihar and Sanjesh Tiwari, respectively.

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Fig. 1. Effect of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) in As1 (6 mg kg-1 soil) and

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As2 (12 mg kg-1 soil) stressed Helianthus annuus L. seedlings on the root fresh mass (RFM),

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shoot fresh mass (SFM), root length (RL), shoot length (SL) and leaf area (LA). Data are

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means±standard error of the three independent experiments. Bars followed by the same letter are not significantly different according to DMRT at P<0.05 significance level.

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Fig. 2. Arsenic accumulation in different plant parts during As1 (6 mg kg-1 soil) and As2 (12 mg kg-1 soil) alone toxicity and in combination with of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6

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kJ m-2d-1) in Helianthus annuus L. seedlings. Data are means±standard error of the three independent experiments. Bars followed by the same letter are not significantly different

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according to DMRT at P<0.05 significance level.

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Fig. 3. Effect of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) in As1 (6 mg kg-1 soil) and

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As2 (12 mg kg-1 soil) stressed Helianthus annuus L. seedlings on chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids (Car), anthocyanin and flavonoids. Data are means±standard error of the three independent experiments. Bars followed by the same letter

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are not significantly different according to DMRT at P<0.05 significance level. Fig. 4. Effect of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) in As1 (6 mg kg-1 soil) and As2 (12 mg kg-1 soil) stressed Helianthus annuus L. seedlings on net photosynthetic rate and respiration rate. Data are means±standard error of the three independent experiments. Bars followed by the same letter(s) are not significantly different according to DMRT at P<0.05 significance level. Fig. 5. Effect of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) in As1 (6 mg kg-1 soil) and As2 (12 mg kg-1 soil) stressed Helianthus annuus L. seedlings on superoxide radical (SOR, O2•¯), H2O2, malondialdehyde (MDA), reactive carbonyl group (RCG) generation and

ACCEPTED MANUSCRIPT electrolyte leakage (EL) in Helianthus annuus. Data are means±standard error of the three independent experiments. Bars followed by the same letter(s) are not significantly different

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according to DMRT at P<0.05 significance level.

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Fig. 6.Effect of UV-B1 (1.2 kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) in As1 (6 mg kg-1 soil) and

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As2 (12 mg kg-1 soil) stressed Helianthus annuus L. seedlings on superoxide dismutase (SOD), POD (POD), catalase (CAT), glutathione-S-transferase (GST) and proline (Pro). Data

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are means±standard error of the three independent experiments. Bars followed by the same

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TE

D

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letter are not significantly different according to DMRT at P<0.05 significance level.

ACCEPTED MANUSCRIPT

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D

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Graphical abstract

ACCEPTED MANUSCRIPT Table 1: Effect of UV-B1 (1.2

annuus L. seedlings under As1 (6 mg kg-1 soil) and As2 (12 mg kg-1 soil).

Treatments

Photosynthetic electron transport rate [µmol O2 evolved/ consumed (mg Chl a h)-1] PS I a

136.00±3.93

(100.00%)

121.00±3.49

b

124.00±3.58b

As1+ UV-

(91.18%) 106.00±3.06

cd

(77.94%)

B1 As2+ UV-

95.20±2.75

e

(70.00 %)

D

B1 UV-B2

(85.00%)

96.60±2.79

As2+ UV-

de

(71.03%)

CE P

As1+ UV-

TE

115.60±3.34bc

81.60±2.36 (60.00%)

ab

(100%)

86.00±2.48b (86.00%) 77.00±2.22c (77.00%)

258.00±7.45a

88.00±2.54b

(96.99%)

(88.00%)

247.00±7.13

ab

(92.86%)

229.00±6.61

73.00±2.11c (73.00%)

bc

66.00±1.91d

(86.09%)

(66.00%)

253.00±7.30a

79.00±2.28c

(95.11%)

(79.00%)

223.00±6.44

c

(83.83%) f

100.00±2.89a

(93.98%)

MA

UV-B1

250.00±7.22

NU

c

(80.15 %)

As2

a

(97.74%)

109.00±3.15

B2

260.00±7.51

(88.97 %)

As1

B2

266.00±7.68

SC R

(100%)

As0

Whole chain

a

IP

PS II

T

activities in Helianthus

kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) on photosynthetic electron transport

213.00±6.15 (80.08%)

63.00±1.82d (63.00%)

c

52.00±1.50e (52.00%)

Data are means±standard error of three independent experiments. Values with different letter(s) within the same

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column show significant differences at P<0.05 level between treatments according to the Duncan’s Multiple Range Test. Values in parentheses indicate readings at per cent scale considering control as 100%.

ACCEPTED MANUSCRIPT Table 2: Restoration of PS II activity by DPC, NH2OH and MnCl2 in exposed to UV-B1 (1.2

Helianthus annuus L. seedlings

kJ m-2d-1) and UV-B2 (3.6 kJ m-2d-1) under As1 (6 mg kg-1 soil) and As2 (12 mg kg-1

soil).

105.00±3.03

As0

94.50±2.73

DPC

As2

114.80±3.31

104.90±3.03b

112.20±3.24cd

135.00±3.90b

93.50±2.70cd

(85.00%)

(90.00%)

(82.02%)

e

103.00±2.97

D

cd

(85.05%)

68.30±1.97

59.90±1.73 (57.05%)

d

(78.03%)

CE P

As2+ UV-

(85.00%) ab

84.00±2.42de

f

(65.05%)

B2

96.90±2.80bc

(92.02%)

89.30±2.58b

As1+ UV-

104.90±3.03b

(96.00%)

TE

UV-B2

144.00±4.16

(100.00%)

(93.03%)

(73.05%)

B1

135.00±3.90

NU

122.80±3.54

114.00±3.29a

(92.02%) b

(90.00%)

ab

MnCl2

(91.05%)

76.70±2.21

As2+ UV-

bc

(86.97%) b

(80.00%)

B1

145.50±4.20

ab

(97.00%)

MA

As1+ UV-

ab

(93.94%) cd

95.60±2.76

150.00±4.33

a

(100.00%)

124.00±3.58

(83.05%)

UV-B1

a

(100.00%)

bc

(90.00%) 87.20±2.52

NH2OH

132.00±3.81

(100.00%)

As1

B2

a

IP

Without donor

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Treatments

T

PS II activity [µmol DCPIP photoreduced (mg Chl a h)-1]

g

116.20±3.35

(81.00%) bc

(88.03%)

92.40±2.67

(64.02%)

139.50±4.03

109.50±3.16

102.00±2.94 (68.00%)

99.20±2.86bc (87.02%)

d

(73.00%) e

86.60±2.50d (75.96%)

ab

(93.00%) e

(70.00%) 84.50±2.44

121.50±3.51

c

75.50±2.18e (66.23%)

d

68.40±1.97e (60.00%)

Data are means±standard error of three independent experiments. Values with different letter(s) within the same

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column show significant differences at P<0.05 level between treatments according to the Duncan’s Multiple Range Test. Values in parentheses indicate readings at per cent scale considering control as 100%.

ACCEPTED MANUSCRIPT

[UVB+Arsenic ]

Root fresh 22.578 mass F <0.00 P- value 1

48.362 <0.001

1.194 0.347

Root Length F P- value

20.795 <0.00 1

33.230 <0.001

3.040 0.044

RootAs content F P- value

49.850 <0.00 1

1.790 <0.001

13.056 <0.001

57.244 <0.00 1

1.808 <0.001

20.444 <0.001

12.936 <0.00 1

14.490 <0.001

0.389 0.813

Chl b F P- value

29.303 <0.00 1

13.557 <0.001

24.961 <0.001

Anthocyanin F P- value

51.679 <0.00 1

18.538 <0.001

0.103 0.980

4.127 0.0330

4.614 0.240

0.980 0.443

48.540 <0.00 1

54.414 <0.001

3.945 0.018

H2O2 F P- value

55.020 <0.00 1

43.595 <0.001

5.850 0.003

RCG F P- value

Carotenoid F P- value Flavanoids F P- value Respiratio n F P- value SOR F P- value

MDA F P- value

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Shoot Length F P- value

[UVB+Arsenic ]

17.392 <0.00 1

28.321 <0.001

0.276 0.890

16.483 <0.00 1

12.620 <0.001

1.674 0.200

76.01 <0.00 1

1769.00 <0.001

21.16 <0.001

11.211 0.001

12.042 <0.001

0.218 0.925

51.910 <0.00 1

13.248 <0.001

0.083 0.987

62.348 <0.00 1

43.608 <0.001

3.539 0.027

83.443 <0.00 1

67.335 <0.001

9.863 <0.001

54.263 <0.00 1

47.317 <0.001

5.525 0.004

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Shoot fresh mass F P- value

As

MA

Shoot content F P- value

[Arsenic ]

IP

Chl a F P- value

[UVB]

D

Leaf area F P- value

Parameters

T

[Arsenic ]

TE

[UVB]

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Parameter s

of two stressors and the studied parameters in Helianthus annuus

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Table.3 Two-way ANOVA results

Photosynthesi s F P- value

ACCEPTED MANUSCRIPT

POD F P- value

11.876 0.001

8.322 0.003

6.578 0.002

GST F P- value

34.220 <0.00 1

82.866 <0.001

2.927 0.050

74.352 <0.00 1

53.158 <0.001

0.631 0.647

75.560 <0.00 1

56.085 <0.001

3.072 0.043

T

1.428 0.265

IP

195.578 <0.001

MA

NU

SC R

73.417 <0.00 1

D

Proline F P- value

9.677 <0.001

TE

CAT F P- value

106.115 <0.001

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SOD F P- value

61.178 <0.00 1

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EL F P- value

ACCEPTED MANUSCRIPT Highlights

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The interactive effect of UV-B and As on the growth of Helianthus annuus Pinpointed exact mode of action of damage on photosynthetic activity by using electron donors Damaging effect by analysing damage and their indices Arsenic posed more toxicity than UV-B and damage was enhanced under combined stressing Significant interactive effects on studied parameters suggest synergistic effect of twin stressors

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