Phaseolus vulgaris L. Seedlings Exposed to Prometryn Herbicide

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Phaseolus vulgaris L. seedlings exposed to the prometryn herbicide contaminated soil trigger an oxidative stress response kerima boulahia, Pierre Carol, Severine Planchais, and ouzna Abrous-belbachir J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00328 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on April 4, 2016

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Journal of Agricultural and Food Chemistry

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Phaseolus vulgaris L. seedlings exposed to prometryn herbicide contaminated soil

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trigger an oxidative stress response

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Kerima Boulahiaa, Pierre Carolb, Séverine Planchaisb, Ouzna Abrous-Belbachira

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a

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Biologiques. Université des Sciences et de la Technologie Houari Boumediene, BP 32, El

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Alia, Bab Ezzouar, Alger, Algérie.

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b

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Pierre et Marie Curie, Place Jussieu. UPMC-EAC.7180CNRS, 75252 Paris. Cedex. 05.

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France.

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Laboratoire de Biologie et Physiologie des Organismes (LBPO). Faculté des Sciences

Laboratoire Adaptation des Plantes aux Contraintes Environnementales URF5. Université

*E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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Herbicides from the family of S-triazines, such as prometryn, have been widely used in crop

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production and can constitute an environmental pollution both in water and soil. As a valuable

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crop, the common bean (Phaseolus vulgaris L.) is grown all over the world and could be

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exposed to such herbicides. We wanted to investigate the possible stress sustained by the

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common bean growing in prometryn-polluted soil. Two situations were observed: when soil

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was treated with 100 µM prometryn or more, some, but not all, measured growth parameters

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were affected in a dose-dependent manner. Growth was reduced, photosynthetic pigments and

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photosynthetic products were less accumulated when soil was treated with 100 µM prometryn

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or more. Reactive oxygen species (ROS) produced had a deleterious effect, as seen by the

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accumulation of oxidized lipid in the form of malonyldialdehyde (MDA). Higher prometryn

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(500 µM) concentrations had a disastrous effect, reducing antioxidant activities. At a low (10

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µM) concentration, prometryn increased antioxidant enzymatic activities without affecting

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plant growth or MDA production. Gene expression of proline metabolism genes and proline

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accumulation confirm that bean plants respond to a stress according to the prometryn

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concentration. Physiological responses such as antioxidative enzymes APX, CAT and the

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enzyme implicated in the metabolisation of xenobiotics GST were increased at 10 and 100

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µM which indicated a prevention of deleterious effects of prometryn, suggesting that bean is a

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suitable material for both herbicide pollution sensing and as a crop on low-level of herbicide

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pollution.

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KEYWORDS: prometryn herbicide; Bean (Phaseolus vulgaris L); Antioxidant systems;

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Gene expression; Proline.

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1 – INTRODUCTION

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Despite the beneficial effect of herbicides in modern agriculture, especially for managing

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weeds and increasing productivity, its general use can have adverse environmental effects.

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Herbicides can be selective when used at specific doses, however, all herbicide molecules are

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supposed toxic for all of the plants.1 Herbicides generate an abiotic stress which can result in

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reactive oxygen species (ROS) production.2-4 ROS result from oxygen metabolism, they

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include free radicals such as superoxide (O2•-), singlet oxygen (1O2) and hydroxyl radical

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(OH•), and non radical hydrogen peroxide (H2O2).5,6 ROS can react chemically with cell

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components such as lipids, proteins and nucleic acids altering cell function leading to a

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diminished metabolism and cell death.7-9 ROS are produced within the plants cell organelles,

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in plastids, mitochondria and peroxisomes and by membrane associated NADPH-oxidase.10-

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production sites.14,15 ROS are also sensed by the plant cell and can act as a signal molecule

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that can induce ROS-detoxifying responses.16-18 Plant cells have evolved ROS detoxification

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mechanisms.13,19 Chloroplasts,14 mitochondria20 and peroxisomes21 are equipped with ROS

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detoxification enzymes.

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Prometryn [2,4-bis (isopropylamino)-6-(methylthio)-S-triazine], a selective herbicide of the

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S-triazine chemical family, has been utilized for controlling annual grasses in agricultural

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practice.22,23 Prometryn is relatively water soluble, it is applied directly to the soil before

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sowing or pre-germination.22 Prometryn can bind photosystem II (PS II) D1 protein,24-26

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which blocks electron transfer.27 As a consequence of prometryn action, the light-activated

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PSII produce singlet oxygen (1O2), which as a ROS will lead to the destruction of thylakoid

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membranes, photosynthetic pigments and eventually cell and plant death.28-32 At moderate

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concentrations, prometryn can reduce plant growth and generate an oxidative stress.22,33

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Several molecules have non enzymatic antioxidant properties such as reduced glutathione (γ-

In plant cells the chloroplast photosystems I and II (PSI and PSII) are major ROS

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glutamyl-cysteinyl-glycine, GSH) which displays a thiol function that can react with ROS and

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act as a strong reductant.34,35 Ascorbate (Vitamin C) in association with enzyme ascorbate

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peroxidase (APX) is also important, it can reduce H2O2 to H2O in chloroplasts and

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cytosol.36,37 Enzymes such as superoxide dismutase (SOD) catalyze the transformation of the

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superoxide to H2O2 and O2.38,39 The produced H2O2 is in turn converted into H2O and O2 by

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several other antioxidant enzymes, such as catalase (CAT).40,41 SOD and CAT are mainly

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located in mitochondria and glyoxysomes or peroxisomes.40,42,43 CAT activity is of special

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importance because it does not require reducing power to eliminate H2O2.44 If not eliminated,

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H2O2, which is relatively stable and soluble can act at distance from its site of production

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either as a deleterious molecule or as a signal.45,46 Guaiacol peroxidase (POD) is also a

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potential H2O2 scavenger when a wide range of electron donors are available.47,48 Other

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antioxidant molecules effectively protect cell components against ROS. Carotenoids protect

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the photosynthetic apparatus by quenching a triplet sensitizer (Chl3), 1O2 and other harmful

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free radicals naturally formed during photosynthesis.49,50 Tocopherols (Vitamin E) are

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considered general antioxidants for protection of membrane stability, including quenching or

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scavenging ROS, like singlet oxygen 1O2.51,52 Flavonoids are able to lower lipid peroxidation

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and stabilize membranes by decreasing membrane fluidity and ROS diffusion.53,54 The amino

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acid proline which is accumulated during abiotic stress has also been associated to an

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antioxidant effect.55,56 Herbicides as xenobiotic molecules can be eliminated by the

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glutathione S-transferase (GST) enzyme, which uses GSH as a substrate to conjugate with the

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xenobiotic.57,58 Although prometryn is widely used as an herbicide and its action as a S-

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triazine molecule largely understood, less is known on its general effect on cultivated

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plants.22,33 We wanted to elucidate the effect of prometryn on common bean (Phaseolus

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vulgaris L.) as a crop plant. In order to understand the long-term effect of prometryn molecule

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in soil, especially at doses that are similar or lower to that of its herbicidal effect. We

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measured growth and some physiological parameters and several antioxidant activities (APX,

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CAT, GST). Molecules resulting from an herbicidal effect on photosynthesis (chlorophyll,

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soluble sugar) or from ROS lipid peroxidation (malonyldialdehyde: MDA) or proline

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accumulation were analysed. Furthermore gene expression in relation with herbicidal stress

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was also probed.

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2 – MATERIALS AND METHODS

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2.1 - Used herbicide

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Prometryn, (commercially named Gesagard 480SC) was purchased from Syngenta Crop

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Protection at a concentration of 480 g.L-1. The recommended use is 1.5 to 4 L/ha depending

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on the type of soil. The highest concentration used here (100 and 500 µM) are similar to the

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recommended doses.

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2.2 - Plant growth conditions and herbicide treatment

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Common bean (Phaseolus vulgaris L. var. Contender) seeds were kindly supplied by

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Industrial and Vegetable Crops Institute (ITCMI, Algeria). Seeds were surface sterilized with

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diluted sodium hypochlorite (1%) for 3 minutes and then rinsed thoroughly with water. Seed

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germination was induced by incubation on water soaked paper for 3 days at 26°C. Once

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germinated, seedlings were transplanted into plastic containers containing 60 g of a mixture

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peat, perlite and vermiculite (2/1/1; vol/vol/vol). The peat, reference « V1 mix » from Jiffy

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Products International BV (The Netherland) is a mix of white and black peat added

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with fertilizer NFU42001 with a NPK content of 17, 10, 4 kg/m3 respectively. After

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preliminary tests, three herbicide concentrations were chosen: 10 µM, 100 µM and 500 µM.

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The choice of these concentrations was based on the morphological appearance of bean

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seedlings subjected to second stage trifoliate leaf in a concentration range of prometryn.

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Concentrations above 500 µM cause seedling death. The treatment is carried out by watering

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the seeds sown in pots with solutions of increasing concentrations of prometryn: 10 µM (dose

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considered low as involving no apparent morphological effects), 100 µM (similar to the

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recommended field dose) and 500 µM (high dose). Plants were watered once with 25 mL of

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different concentrations of prometryn. Controls were irrigated with 25 mL of water. From

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the second day, plants (Control and treated) grown in a greenhouse under a 16 h light/8h

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dark regime, were regularly watered with an appropriate volume of water.

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2.3- Estimation of growth and dry matter mass

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Length of aerial parts was measured from the base of the stems (crown) to the apical bud for

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21 days. At this stage the first trifoliate leaf is fully developed. Dry matter mass was obtained

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after total desiccation of aerial parts and roots (separated beforehand) for 48 hours at 65 ° C.

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Each data point represents the average of thirty plants.

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2.4- Chlorophyll content

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Total pigments from 10 mg of fresh leaves were extracted in 2 mL of 80% (v/v) acetone.

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Chlorophyll content was determined by spectrophotometric absorbance as recommended by

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Lichtenthaler.59

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2.5- Soluble sugars content

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Soluble carbohydrates content was determined according to the method described by Mc

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Cready et al,60 based on the use of anthrone reagent in sulfuric acid. Extraction of soluble

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sugars is done by grinding 200 mg of fresh leaves in 4 mL of boiling 80 % ethanol. After

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agitation and centrifugation at 5000 rpm for 20 minutes, the sugar containing supernatant was

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recovered. The volume was adjusted to 10 mL with distilled water. 0.5 mL of carbohydrate

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extract was added to 0.5 mL of distilled water and 2 mL of anthrone reagent. Reaction mix

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were homogenized and placed at 100°C for 7 minutes. After cooling, measurements were

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performed by absorbance at 630 nm. Soluble carbohydrate concentration was determined

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using a glucose standard curve.

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2.6- Lipid peroxidation analysis

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Lipid peroxidation was determined by measuring the concentration of malondialdehyde

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(MDA) as thiobarbituric acid reactive substances (TBARS) based on the method described by

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Alia et al.61

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Fresh tissues (100 mg of leaves) were ground and homogenized in 2 mL of 0.1 %

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trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 13000 rpm for 15

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minutes at 4°C and 0.5 mL of the supernatant was mixed with 0.5 mL of 0.5 % thiobarbituric

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acid (TBA) in 20 % TCA. The mixture was boiled in a water bath for 25 minutes, chilled on

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ice, and centrifuged at 10000 rpm for 5 minutes at 4°C. The absorbance of the supernatant (or

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TBA-MDA complex) was measured at 532 nm. The concentration of TBARS was calculated

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by using the extinction coefficient ε = 155 mM-1cm-1.

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2. 7 - Estimation of membrane integrity by electrolytes leakage measurement

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Electrolytes leakage (EL) was measured according to the method of Dionisio-Sese and

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Tobita.62 Fresh leaves were cut into uniform size disks (8 mm diameter) and then placed in

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test tubes containing 20 mL of distilled water. The initial electrical conductivity of the

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medium (EC1) was measured after 60 minutes of incubation of samples at room temperature.

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The samples were thereafter put in a water bath at 90 °C for one hour in order to trigger the

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release of all electrolytes. After cooling, the final electrical conductivity (EC2) was

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determined. Electrolytes Leakage of (EL) was expressed as % of the total amount of the

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electrolytes.

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2.8- Proline extraction and content

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Proline content was determined by a colorimetric method adapted from Bates et al.63 50 mg of

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fresh tissue (from the first leaf of 21 days old plants) were ground in 1.5 mL of 3 %

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sulfosalicylic acid. The homogenate was centrifuged at 14000 rpm at 4°C for 10 minutes. 1

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mL reagent ninhydrin buffer (2.5% ninhydrin, 60 % acetic acid and 2.5M Phosphoric acid)

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and 1 mL of 100 % acetic acid were added to 1 mL of supernatant, and then boiled in water

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bath for 60 minutes. After cooling, 2 mL of toluene was added to each sample. The upper

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organic phase optical density was read at 520 nm. Proline quantification was determined

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using L-proline as standard.

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2.9- Glutathione (GSH) extraction and content

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GSH assay is based on the colorimetric method of Ellman.64 The principle is based on the

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oxidation reaction of GSH with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) thus releasing the

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5-thio-2-nitrobenzoic acid (TNB) absorbing at 412 nm.

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For this assay, 200 mg of fresh leaves or roots were homogenized in three volumes of a 5%

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sulfosalicylic acid and then centrifuged at 13000 rpm at 4°C for 10 minutes. 200 µL of

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supernatant were diluted in 1 mL of 0.2M phosphate buffer (pH 8) and 100 µL of Ellman's

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reagent (0.04 %) prepared in phosphate buffer. The resulting mixture was incubated at room

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temperature for 10 minutes. Absorbance at 412 nm was determined by spectrophotometry.

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The concentrations were deducted from a standard curve established with glutathione.

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2.10- Assays of enzyme activities

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Leaves or roots (around 100 mg fresh mass) were homogenized at 4°C with 1 mL of an ice-

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cold buffer containing 0.1M Tris-HCl buffer (pH 8.1), 10 % of sucrose and 0.05 % of β-

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mercaptoethanol. Extracts were centrifuged at 13000 rpm for 10 minutes at 4°C. The

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supernatant was recovered and was then used for the measurement of various enzyme

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activities. Protein content was determined as described previously by Bradford65 using Biorad

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protein assay reagents with BSA as a standard. 8


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Ascorbate peroxidase activity

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Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by measuring the

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decrease in the concentration of ascorbate at 290 nm (ε = 2.88 mM-1 cm-1) during the

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reaction.66 The reaction mixture was composed of 50 mM potassium-phosphate buffer pH 7.5,

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0.1 mM EDTA and 30 µg of protein extract. The reaction was initiated by adding

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subsequently 10 µL of ascorbate to 0.5 mM and 5 µL of H2O2 to 0.06 % (corresponding to 0.1

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mM). The decrease of absorbance at 290 nm was measured at 30 seconds and 1 minute. The

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enzymatic activity was expressed in nanomoles of ascorbate oxidized per minute and per mg

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of protein.

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Catalase activity

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Degradation of H2O2 by catalase (CAT, EC 1.11.1.6) was followed directly by the decrease in

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absorbance at 240 nm (H2O2molar extinction coefficient ε = 36 M-1.cm-1).67 A volume

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corresponding to 30 µg of protein was placed in a reaction medium (1 mL) containing 50 mM

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sodium phosphate buffer (pH 7.5). The reaction was triggered by the addition of 5 µL 6 %

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H2O2 (corresponding to 10 mM). The variation of absorbance was followed every 30 seconds.

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The CAT activity was expressed in nanomoles of H2O2 degraded per minute and per mg of

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protein.

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Glutathione S-transferase activity

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Glutathione S-transferase (GST, EC 2.5.1.18) activity was performed according to the

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protocol adopted by Habig et al68 based on the reaction of conjugation between GST and

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CDNB (1-chloro-2, 4-dinitrobenzene) in the presence of glutathione (GSH). This activity was

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measured at 340 nm and a coefficient of molar extinction ε = 9.6 M-1.cm-1. Leaves or roots

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(500 mg) samples were crushed cold then homogenized in 500 µL Tris-EDTA (10 mM Tris, 1

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mM EDTA, pH 7.8) and a few drops of an antioxidant Polyvinylpolypyrrolidone (PVPP) 50

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mg.mL-1. After incubation in ice for 15 minutes, the resulting homogenate was centrifuged at

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13000 rpm and 4°C for 15 minutes. The supernatant will serve as source of enzyme. The final

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reaction volume contained 100 µL of the enzyme extract (approximately 60 µg protein), 30

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µL of CDNB to 100 mM and 3 mL of 0.1M phosphate buffer (pH 6.5). The addition of 30 µL

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100 mM glutathione (GSH) started the reaction. Values were recorded every 15 or 30 seconds

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depending on the speed of the reaction. The enzymatic activity was expressed in nanomoles

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per minute and per mg of protein.

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2.11- Statistical analysis

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The results are given as averages and standard deviations of at least ten replicates (except for

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growth assays, 30 plants were used), obtained from three independent experiments. The

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statistical evaluation was performed by the analysis of variance (ANOVA).The comparison of

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values obtained from treated plants compared with the control plants was performed using the

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Student's t-test. This test was used when the samples were compared in pairs. The probability

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given by the t calculation allowed us to assess the significance of the difference between

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control and treated samples.

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P < 0.05 = significant difference (a). P < 0.01 = very significant difference (b). P < 0.001 =

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highly significant difference (c).

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2.12- Gene expression analysis

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RNA extraction

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100 mg of ground leaves were homogenized in RNA extraction buffer (0.2M Tris-HCl,

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pH7.5, 0.25M NaCl, 25mM EDTA, 0.5% SDS) and mixed with an equal volume of citrate-

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buffered (pH=4) phenol:chloroform (1:1, vol/vol). Samples were centrifuged at 10000 rpm for

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5 minutes. The upper aqueous phase was recovered and re-extracted and RNA was selectively

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precipitated with 2M LiCl at 0°C overnight. RNA was pelleted by centrifugation at 12000

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rpm for 15 minutes and dissolved in water. A second precipitation of RNA with 2M LiCl was

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done for 6 hours at 0°C. After centrifugation, RNA pellets was washed with 70 % ethanol,

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dried and dissolve in 50 µL water. RNA was quantified by UV (260 and 280 nm) absorbance

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using a Nanovue spectrophotometer. RNA integrity was checked by electrophoresis on 1%

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agarose gel.

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RT-PCR

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1.5 µg of RNA was used to perform reverse transcription with Revert Aid Reverse

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Transcriptase according to the manufacturer’s instructions (Thermo Scientific). cDNAs

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samples were diluted 4 times with ultrapure water.

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PCR was done using Dream Taq Green DNA polymerase (Thermo Scientific). For each PCR

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reaction, 2 µL of cDNA was used as a template, 0.8 µM of forward and reverse primers, 0.2

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mM dNTP, 1 unit of DreamTaq in 1X GreenTaq Buffer. PCR conditions were: 5 min at

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94°C, followed by a cycle of 30s at 94°C, 30s at 55°C and 30s at 72°C repeated 28 to 32

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times according to each gene expression followed by 10 minutes at 72°C. Primers

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characteristics are described in Table 1.

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3 - RESULTS

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Bean Plants Growth is inhibited on soil soaked with increasing concentrations of

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prometryn.

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In order to quantify the possible effect of prometryn, we grew germinated bean seedlings in

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soil substrate soaked with increasing concentrations of prometryn solutions (10, 100 and 500

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µM). We measured plant growth over 21 days and compared control plants with plants

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growing in prometryn-polluted soils (Figure 1A). Over time, control plants reached an

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average 29 (± 1.61) cm and developed the first trifoliated leaves.

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The lowest dose of prometryn (10 µM) did not affect plant growth significantly. However, at

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higher doses, prometryn had a severe effect on plant growth. Plants were 26 % shorter when

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grown on 100 µM prometryn and 38 % shorter when grown at the highest dose of 500 µM.

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This effect on plant growth was also observed when measuring the aerial part (shoot and 11



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leaves) dry weight (DW), which was 0.33 g for control plants (Figure 1B). Plants grown on

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the highest prometryn concentrations had 36 % and 51 % lower DW for 100 and 500 µM

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respectively. Roots DW was also similarly affected by prometryn (Figure 1B). A reduction of

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51 % of root DW was observed when plants were grown on the highest concentration of

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prometryn (500 µM).

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As a whole, prometryn at concentration between 100 and 500 µM induced concurrently a

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decrease in size and dry weight corresponding probably to a perturbation in the energetic

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metabolism.

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Content of chlorophyll and soluble sugars is lower in plants exposed to prometryn.

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Prometryn is known to act at the level of the electron transfer chain in the chloroplast, where

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it could damage photosynthesis. Twenty one days after treatment, we quantified

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photosynthetic pigments which were lowered in the same way as the dry weight. From 3.95

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mg.g-1 FW chlorophyll in control plants, chlorophyll content is 36 % and 49 % less in plants

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grown on 100 µM and 500 µM prometryn respectively. Plants growing on the lowest (10 µM)

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have a chlorophyll content similar to that of the control (Figure 2A).

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Concurrently, we found that plants grown in 100 µM and 500 µM prometryn-treated soil

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accumulate less than 40 % soluble sugar to that of control plants (0.38 and 0.28 mg.g-1 FW

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compared to 1.01 mg.g-1 FW in control plants) (Figure 2B). The lowest dose of prometryn in

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soil (10 µM) did not affect significantly sugar content.

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Prometryn induces oxidative damages to lipids.

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As an herbicide that blocks chloroplast electron transfer chain (ETC) at the level of PSII,

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prometryn can induce under light the production of singlet oxygen and other ROS. ROS in

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turn can oxidize lipids and produce MDA. We measured MDA as an indication of ROS action

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in leaf cells of bean plants (Figure 3A). The basal level of MDA (24 nmol. g-1) increased 81

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% with 100 µM prometryn and up to 148 % for the highest dose (500 µM). At lowest dose

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(10 µM) of prometryn there was no significant difference with the control (Figure 3A).

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A consequence of lipid oxidation by ROS is a loss of membrane integrity, which can have

280

potential harmful consequences for cell metabolism leading to leakage of small molecules

281

such as electrolytes. Electrolytes leakage for control plants is of 32 % (Figure 3B). A

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significant increase in electrolytes leakage was observed in relation with the dose of

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prometryn in the soil. Leakage was 39 %, which is close to the control, for 10 µM prometryn.

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Higher leakage values, 47 % and 50 %, were measured for 100 µM and 500 µM prometryn.

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There is therefore a correlation between ROS-generated MDA (about 78 %) and loss of

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membrane integrity (45 %) in bean plants subjected to a continuous exposure to prometryn.

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Prometryn modifies non enzymatic antioxidant level.

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Reduced glutathione (GSH) is a key component of antioxidant defenses in most aerobic

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organisms.70 GSH content in roots and leaves is lower for a prometryn exposure of 10 and

290

100 µM (Figure 4). The lowest GSH content is measured in plants growing on 100 µM

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prometryn both in leaves (44.6 % percent relative to controls) and in roots (56.8 % percent

292

relative to controls). However, the highest prometryn concentration (500 µM) resulted in a

293

higher GSH content both in leaves (31 % more) and roots (8.6 % more).

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Prometryn induces changes in antioxidant enzymatic activity.

295

ROS can be detoxified by anti-oxidative enzymes, such as ascorbate peroxydase (APX),

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catalase (CAT) and gluthatione S-transferase (GST). We measured these enzymatic activities

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from roots and leaves after 21 days culture. A general trend to our measurements is that bean

298

plants growing in prometryn-containing soil display enzymatic activities different to that of

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the control plants (Figure 5).

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In control plants APX activity is three times higher in leaves (715 nmol.min-1.mg-1) than in

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roots (248 nmol. min-1 mg-1). Despite this three-time difference, APX activity increases both

302

in leaves and roots from the lowest dose (10 µM) and for 100 µM of prometryn, with

303

increases up to 42 % in leaves and 64 % in roots (Figure 5A). However the highest prometryn

304

dose leads to a lower than control APX activity with a significant 49 % decrease in APX

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activity in leaves and 22 % decrease in roots compared to the control (Figure 5A).

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CAT activity is more than fourfold higher in leaves compared to roots (84 nmol.min-1.mg-

307

1

308

increasing doses of prometryn. For 100 µM prometryn, CAT increased over 100 % in leaves

309

and increased up to 77 % in roots. CAT however decreased for the highest dose of 500 µM

310

prometryn with a 25 % lower level than in the control (Figure 5B).

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GST activity is quite similar in leaves and roots (46 nmol.min-1.mg-1) (Figure 5C). GST

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activity in leaves increased with prometryn and was 2.5 higher in plants growing on 100 µM

313

prometryn. As seen with other antioxidant activities, GST activity was lower than control in

314

plants growing on the highest prometryn concentration (500 µM). In roots variation of GST

315

activity followed a similar trend with a noticeable increase for the 10 and 100 µM

316

concentrations and decreased at the highest concentration. The variations were however more

317

modest 16 % higher at 100 µM than the control or 11 % lower at 500 µM (Figure 5C).

318

Bean plants grown on 10 to 100 µM of soil-prometryn displayed a significant decrease of

319

GSH but an increase in enzymatic (APX, CAT and GST) activities. This suggests an active

320

anti-oxidative enzymatic activity corresponding to GSH consumption.

321

Plants grown on the highest (500 µM) prometryn concentration displayed a significant

322

increase in GSH but a decrease in all three tested enzymatic activities. This could reflect a

323

general disfunctioning of the plant probably inducing death at this concentration.

and 16 nmol.min-1.mg-1 respectively) (Figure 5B). CAT activity increased in leaves with

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324

Because prometryn oxidative stress occurs in the chloroplast it can affect the overall Red-Ox

325

balance. We tested the possible consequences of the oxidative stress on genes that are usually

326

target of Red-Ox regulation. Alternative oxidases, from the mitochondria (AOX) or from the

327

chloroplast (PTOX) had been shown to be induced by stress and, in some cases, to be

328

involved in lowering ETC induced ROS production.71,72 We tested AOX and PTOX gene

329

expression using RT-PCR. We found they were similarly expressed whatever the prometryn

330

concentration in soil or the leaf tested (Figure 6), suggesting that the oxidative stress triggered

331

by prometryn does not modulate these genes.

332

Prometryn-induced stress triggers proline accumulation and proline metabolism genes

333

expression.

334

Proline is an amino acid, which accumulates in many plant species in response to stress and to

335

ROS signalling.73 We reasoned that bean plants growing in prometryn-treated soil showing

336

oxidative stress damages could also accumulate proline. We measured free proline

337

accumulation in leaves from treated and control plants (Figure 7). Control plants accumulate

338

very little free proline (26.61 µg.g-1 FW). Plants growing on the lowest dose of prometryn did

339

not show any significant proline accumulation. However, on higher doses of herbicide (100

340

µM and 500 µM) plants accumulated over a threefold more proline (78.06 and 95.32 µg.g-1

341

FW) (Figure 7).

342

We tested the possible regulation of proline metabolism at the gene expression level. Stress

343

can up-regulate the P5CS gene encoding the enzyme delta 1-pyrroline-5-carboxylate

344

synthetase.74,75 Stress can also regulate (up or down) proline catabolism gene PRODH,

345

encoding proline dehydrogenase (ProDH).74 Proline metabolism genes were scarcely

346

expressed in control plants but their expression increased in plants exposed to prometryn

347

(Figure 8). P5CS mRNA accumulation increased the most dramatically in plants exposed to10

15



Page 16 of 45

348

µM prometryn. P5CS expression was not fully correlated with proline accumulation here.

349

PRODH mRNA accumulation increased mostly in plant treated with the highest concentration

350

of prometryn (500 µM) suggesting that P5CS and PRODH were differently regulated, being

351

responsive to different levels of stress. PRODH gene expression could antagonise proline

352

accumulation in plants treated with the highest dose of prometryn however it was not

353

correlated with a lower proline accumulation but with a modest increase in proline

354

accumulation when comparing plants exposed to 10 µM and 500 µM prometryn (Figure 7).

355

4 - DISCUSSION

356

Prometryn is an S-triazine herbicide which acts as an inhibitor of chloroplast electron transfer

357

chain (ETC) at the level of photosystem II (PSII)25,76,77 and affects as a consequence, Rubisco

358

activity, in plants such as in faba bean (Vicia faba).78 However C4 plants such as maize (Zea

359

mays) display a relative tolerance to such molecules.78,79

360

It is not surprising that synthetic herbicides molecules can impair growth80,81 although the

361

main biochemical effect of these different herbicide families was not the same.

362

We found that prometryn has an overall negative effect on bean growing on culture chamber

363

in soils contaminated with 100 µM or more prometryn. Other plant species, such as wheat22,33

364

and pea82 growing in the presence of prometryn also display a growth reduction. Furthermore,

365

herbicides from the same chemical family show also a growth reducing effect. Such

366

molecules include atrazine,39,83 terbutryne and simazine82 or metribuzine.84 Growth reducing

367

effect of other herbicides from the urea family is also well documented, such as for

368

chlortoluron,85 isoproturon.86,87 Similarly, diazine-type molecules such as bentazone,86

369

dinitroanilines such as pendimethalin,88 and sulfonylureas such as chlorimuron-ethyl89 also

370

have a growth-reducing effect on plants. Herbicides are accumulated in plant tissues over

371

time, as seen on rice plants (Oryza sativa L) growing in soil contaminated with atrazine.39 We

372

found bean plants have a reduced content of photosynthetic pigment (at and above the 16


Page 17 of 45


373

concentration of 100 µM prometryn). A lower concentration of photosynthetic pigment is

374

observed in many herbicide treated plants.22,39,90 Interestingly, herbicides, which primarily

375

inhibit pigment biosynthesis, such as the pyridazinone family R-4024491 or norflurazon79 also

376

have a growth-reducing effect. Also, chlorophyll degradation or biosynthesis inhibition has a

377

negative consequence on plant growth.4,85

378

It is therefore possible that the effect of prometryn on PSII and photosynthesis or an indirect

379

effect could lead to the diminution of chlorophyll and a slower growth of bean plants.

380

We found that soluble sugars were less accumulated in bean plants growing in soils

381

contaminated with 100 µM and 500 µM prometryn. This could be explained by a lower

382

photosynthesis and less CO2 assimilation and less sugar synthesis. Similar results were

383

obtained in maize treated with atrazine, linuron, prometryn and pyrazon,92 wheat treated with

384

isoproturon93 and Vitis vinifera L. (Vine) stressed with flumioxazin.94

385

Although in our study we correlate exposure to the photosynthesis inhibitor prometryn with

386

lower sugar accumulation, in other cases, sugar has been seen to accumulate, as a

387

consequence of an overall herbicide-stress response. Such examples of sugar accumulation in

388

herbicide-treated plants include wheat plants exposed to chlorotoluron, also a PSII inhibitor.85

389

Also, sugar increases in green algae Chlamydomonas mexicana treated with atrazine,95

390

Chlamydomonas vulgaris and Scenedesmus acutus treated with simazine, another triazine

391

derivative.96 Sugar accumulates in maize treated with protein and DNA synthesis inhibitor

392

imazapyr97 and Thlaspi arvense stressed with amino acid synthesis inhibitor chlorsulfuron.98

393

Sugar accumulation has been observed in response to several other abiotic stresses including

394

water stress and cold stress99,100 with a possible role in cell protection.101,102 Interestingly in

395

Arabidopsis, externally added sugars such as glucose and sucrose seem to protect from the

396

growth inhibitory effect of atrazine.30,103

17



Page 18 of 45

397

As seen before in other plant species we can deduce that bean plants growing in prometryn-

398

contaminated soil have a less effective PSII and are therefore impaired in their

399

photosynthesis, in their CO2 assimilation and display reduced growth.

400

We found that the highest doses of prometryn used in our study (100 µM and 500 µM) lead to

401

MDA accumulation. We therefore measured electrolytes leakage as a consequence of cellular

402

damages occurring in bean plants. It is also possible that the measured lowering in

403

chlorophyll content might result from damage to the thylakoid membrane.

404

Interestingly the lowest dose of prometryn used in our experiment (10 µM) did not lead to

405

growth reduction nor to less sugar content. It did not either increase MDA accumulation.

406

Lipid peroxidation does not occur at the lowest dose of prometryn either because of the low or

407

absent production of ROS or an effective ROS detoxification.

408

Our results are in accordance with other studies regarding prometryn leading to MDA, either

409

in cultivated or in wild species.22,32,33 Other S-triazines types herbicides also lead to MDA

410

accumulation: atrazine for example, in faba bean,78 pea,83 rice39 and maize.31 Abiotic stress

411

also can lead to oxidative damages and MDA accumulation. Such stress includes salt-

412

stress,104 water stress,105 UV radiations,106 high temperature105 and heavy metals.107

413

APX, CAT and GST activity from leaves and roots increases in plants at 10 µM and 100 µM

414

suggesting an activation of enzymatic detoxification mechanisms. Activities were low at the

415

highest dose (500 µM) suggesting either less ROS or an overload of ROS damages. We can

416

rule out the hypothesis that plants treated with the highest dose of prometryn stop making

417

ROS because MDA is most abundant (59.89 ± 7.63 nmole. g-1). Antioxidative enzymes and

418

glutathione S-transferase ability to conjugate prometryn might partly explain the relative

419

resistance of bean to this herbicide. The stimulation of GST may also explain the decrease of

420

reduced glutathione that was observed here. Similar results were observed on wheat where the

421

highest dose of prometryn (20 and 24 mg kg-1) result in a lower enzyme APX and CAT 18


Page 19 of 45


422

activity.25 This has been correlated to an inhibition of CAT activity by elevated dose of

423

H2O2.108

424

Plants grown on a 10 µM prometryn, display a normal growth and sugar and MDA content

425

that are not significantly different to that of the control. Either no significant amount of ROS

426

is produced compared to the control or that detoxification is efficient. Our data suggest that

427

this low dose of prometryn triggers ROS production, which in turn acts as a signal that could

428

increase ROS-detoxifying enzymatic activity at a level sufficient for effectively preventing

429

ROS damages on lipids. Interestingly in Arabidopsis when PSII is blocked by atrazine it leads

430

to less peroxide radical, which could be the direct consequence singlet oxygen production a

431

lower ETC activity and to an increase detoxification in H2O2.44 Stress does induce ROS and

432

ROS-signaling.9,12 Specifically H2O2 and

433

production.22,109

434

We found that proline accumulates in bean growing on prometryn. Proline is accumulated in

435

many plant species in response to abiotic stress such as water stress, salt stress and oxidative

436

stress.73,74,110 Proline-metabolism genes P5CS and PRODH are also responsive to prometryn.

437

When P5CS gene is induced, it allows the first and limiting step of proline biosynthesis from

438

glutamate. P5CS is induced by prometryn from the lowest dose of prometryn, prior to proline

439

accumulation. PRODH which encodes a proline catabolic enzyme is also induced but at high

440

prometryn concentration. Altogether it suggests that the oxidative stress induced by

441

prometryn induces proline metabolism gene expression in bean plants. There is not a clear

442

correlation between PRODH gene expression and proline accumulation suggesting that

443

proline catabolism is not fully operating despite the expression of PRODH.

444

It has been found that the herbicide chlortoluron, also a PSII inhibitor, induces proline

445

accumulation in wheat85 other ROS-producing herbicides produce similar results in a variety

446

of plants species.61,111,112

1

O.2 can activate anti-oxidative enzymes

19



Page 20 of 45

447

Proline itself might be an anti-oxidative molecule and a protective agent as well as a source of

448

carbon and nitrogen in species that accumulate it at high concentrations.113,114,115 In the case

449

of the present study, proline accumulation and proline metabolism gene expression reflect the

450

ROS-induced oxidative stress response.

451

In conclusion, our results demonstrate with our culture conditions, that prometryn at high

452

concentration 100 and 500 µM induces what we named pre-lethal physiological state (Figure

453

9). Both growth and biochemical parameters are profoundly affected, leading to an adverse

454

effect on all mechanisms of detoxification.The main effect is certainly membrane destruction

455

leading to MDA accumulation and on which chloroplasts membranes on the aerial parts are a

456

first target due to ROS emitted under light when the electron transport is inhibited.

457

The decreases in shoot and root elongation, dry weight accumulation, chlorophyll synthesis,

458

enzyme activities can probably be considered as a secondary effect of this partial

459

membranes destruction.

460

One biochemical target seems to be favoured by these drastic conditions, it is the stimulation

461

of PRODH gene transcription; this point requires to be further investigated.

462

Prometryn at 10 µM (figure 10) considered as non-lethal concentration seems not to be

463

detrimental to plant growth, but triggers an oxidative stress and a specific plant response. This

464

concentration might correspond to prometryn remaining in soil months after herbicidal

465

treatment at the recommended dose of 1.5 to 4L/ha. We assume that the low dose prometryn

466

plays a role of a safener inducing signaling mechanisms allowing the plant not to suffer major

467

damage.

468

All the effects summarized, demonstrate the induction of general stress metabolism in bean

469

seedlings, due to prometryn and giving a pattern of effects comparable to those induced by

470

water and cold stresses or by a large panel of other synthetic substances.

20


Page 21 of 45


471

The biochemical responses to prometryn could serve as useful indicators for the assessment of

472

herbicide contamination in agricultural lands.

473

Moreover, the impact of xenobiotics on plants is dependent on xenobiotic chemical structure,

474

on plant physiological status and on plant genotype, thus strongly indicating that gene

475

expression regulations must be integrated with mechanisms of xenobiotic sensing.116

476

ABBREVIATIONS USED

477

ROS, Reactive oxygen species; H2O2, hydrogen peroxide; O2•-,superoxide radical; 1O2, singlet

478

oxygen; OH-, hydroxyl radical; APX, ascorbate peroxidase; CAT, catalase; GSH, glutathione;

479

GST, Glutathione S-transferase; POD, Guaiacol peroxidase; SOD, superoxide dismutase; FW,

480

Fresh Weight; MDA, malonyldialdehyde; AOX, alternative oxidase ; EF1α, Elongation

481

factor; PRODH, proline dehydrogenase ; PTOX, plastid alternative oxidase; P5CS, Pyrroline-

482

5-carboxylate synthase.

21



Page 22 of 45

483

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770

Table 1: Characteristics of Primers.

771

Figure 1: Effects of prometryn on the bean growth. Bean seedlings were cultured in soils

772

containing prometryn at 10 µM, 100 µM and 500 µM for 21 days. Then, the elongation (A)

773

and biomass (B) of both shoots and roots were determined respectively. Values are the means

774

± SD (n = 30).The results were statistically analyzed by Anova followed by t test. Letters

775

indicate the significant differences between the treatments and the control: c highly significant

776

difference (P < 0.001).

777

Figure 2: Effects of prometryn on the content of total chlorophyll (A) and soluble sugar (B)in

778

bean plants. Seedlings were cultured in soils containing prometryn at 10 µM, 100 µM and

779

500µM for 21days, respectively. Then, the contents of chlorophyll and soluble sugar were

780

measured. Values are the means ± SD (n = 10). The results were statistically analyzed by

781

Anova followed by t test. Letters indicate the significant differences between the treatments

782

and the control: c highly significant difference (P < 0.001).

783

Figure 3: Effects of prometryn on the content of TBARS (A) and on electrolytes leakage (B)

784

in bean plants. Seedlings were cultured in soils containing prometryn at 10µM, 100 µM and

785

500 µM for 21days, respectively. Then, the contents of TBARS and electrolyte leakage were

786

measured. Values are the means ± SD (n = 10). The results were statistically analyzed by

787

Anova followed by t test. Letters indicate the significant differences between the treatments

788

and the control: c highly significant difference (P < 0.001).

789

Figure 4: Effects of prometryn on the content of glutathione (GSH) in bean plants. Seedlings

790

were cultured in soils containing prometryn at 10µM, 100 µM and 500 µM for 21days,

791

respectively. Then, the content of glutathione was measured. Values are the means ± SD (n =

792

10). The results were statistically analyzed by Anova followed by t test. Letters indicate the

35



Page 36 of 45

793

significant differences between the treatments and the control: a significantly different (P

Phaseolus vulgaris L. Seedlings Exposed to Prometryn Herbicide

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