Toxicity of Sulcotrione and Grape Marc on Vicia faba CellsChaima Sta,†,‡,§ Eric Goujon,†,‡ Ezzeddine Ferjani,§ and Gerard Ledoigt*,†,‡
†Clermont Universite, Universite Blaise Pascal, UMR 547 PIAF, B.P. 10448, F-63000 Clermont-Ferrand, France‡Campus Universitaire des Cezeaux, 24 Avenue des Landais, 63177 Aubiere cedex, France§Laboratoire de Physiologie et Genetique des Plantes a Interet Agronomique, Faculte des Sciences de Bizerte, Universite de Carthage,7021 Jarzouna, Tunisia
ABSTRACT: The cell toxicity of sulcotrione, a selective triketone herbicide, was evaluated on Vicia faba. Sulcotrione, trademarkMikado, grape marc, and mixtures of sulcotrione or Mikado with grape marc induced cell death. Addition of grape marc to eithersulcotrione or Mikado enhanced cell death, especially with Mikado. Addition of grape marc to herbicides, sulcotrione, or Mikadoresulted in different expression of genes usually associated with cell stress. Mixtures of grape marc and herbicides enhancedtranscript accumulation for ubiquitin, hsp 70, and cytosolic superoxide dismutase, but did not change ascorbate peroxidasetranscript accumulation. The results thus provide evidence that sulcotrione, Mikado, and mixtures with grape marc can triggercell death and specific gene expressions. Cocktails of products with sulcotrione, such as commercial additives and grape marc, canmodify biological features of pesticide. Moreover, grape marc differently enhanced cell toxicity of sulcotrione and Mikado,suggesting a synergy between pesticide products and grape marc.
Conventional pest management has been significantlyinfluenced by bioactive natural products.1 Mikado (BayerCrop Science) is a systemic foliar-applied postemergenceherbicide mostly used for corn crops to control broadleafweeds, such as dicotyledonous plants, and annual grasses.2
Mikado is a trademark for a suspension of sulcotrione (300 g/L) and other compounds not described, used at a rate of 1−2L/ha in fields. In culture, maize population density was 100,000plants per hectare, and application rate amounts were up to 300g of sulcotrione/ha, which yields 3 mg of active substance perplant (or 30 mg/m2).3 Triketone herbicides inhibit the chain ofphotosynthetic electron transfer, therefore blocking a mecha-nism of energy production in plant.3−5 Sulcotrione vaporpressure value, 5 × 10−6 Pa at 25 °C, indicates a low volatilecompound. In an aqueous medium at 25 °C, sulcotrione wasshown chemically stable with a half-life estimated to be between200 and 400 days depending on the pH.3 EFSA6 reported thatthe half-life for the degradation of sulcotrione ranged from 6 to15 days in water and from 48 to 84 days in the environment.Photolysis showed a half-life of 100 days in a solution (pH 7, 25°C). Sulcotrione is not considered readily biodegradable but ismobile, with a Koc of 36 L/kg, and will not tend to adsorb tosuspended solids and sediment.6,7
Sulcotrione, 2-[2-chloro-(4-methylsulfonyl)benzoyl]-1,3-cy-clohexanedione, can be absorbed by leaves and by root systemand may accumulate in the soil more than a month afterapplication. Water solubility of the product is 165 mg/L at 25°C with a great potential to leach.8−10 It belongs to thetriketone herbicides that inhibit 4-hydroxyphenylpyruvatedioxygenase enzyme (p-HPPD), leading to carotenoid ratedecrease in weeds. Loss of carotenoid and α-tocopherol canresult in photosynthesis inhibition, triggering accumulation ofreactive oxygen species (ROS).2,4 In another biological
pathway, sulcotrione induced chromosomal alterations, indicat-ing potent mutagen effects.5,11 Recent studies have shown thatexposure to sunlight can be one of the most destructive factorsfor pesticides following crop treatment that can generate toxicbyproducts4,12,13 in the environment; it can play a significantrole for pesticide effects on both human health and naturalecosystems.14−17
Grape marc has been patented as a new class ofphotoprotecting agent18 that allows pesticide photodegradationto be reduced.19,20 It was shown to trigger an array of plantdefense responses, making this natural compound a potentialphytosanitary product with an issue for sustainable agricultureand environmentally friendly practices.21 Grape marc is ananthocyanin-rich mixture typically containing about 60% ofpolyphenols, among which 8−25% are anthocyanins, and wasobtained from grape pomace.21,22 Anthocyans belong to thelarge family of flavonoids extensively widespread in plants anddisplay antioxidative capacity.23 Association between anthocya-nins and oxidative stress can result in anthocyanin ability toincrease photoprotection and, hence, to reduce a putativeoxidative damage.24,25
Vicia faba is a dicotyledonous plant species that showed agreat susceptibility to clastogenic and cytotoxic chemicals26,27
and belongs to a plant family susceptible to Mikado accordingto Bayer Crop Science. It was therefore used as a target plantmodel for sulcotrione treatments. Earlier papers describeddifferent effects on plants using active molecule or thetrademark product.28,29 Therefore, we have compared treat-ments using either the active ingredient sulcotrione or the
Received: July 14, 2014Revised: October 8, 2014Accepted: October 21, 2014Published: October 21, 2014
commercial product Mikado. The aim of the work was toevaluate consequences of treatments by sulcotrione andtrademark Mikado on plant cell physiology and the ability ofgrape marc to control plant response to determine if suchtreatments can modify pesticide effects on stress metabolism.
■ MATERIALS AND METHODSPlant Material and Chemicals. Sulcotrione, 2-(2-chloro-4-
(methylsulfonyl) benzoyl)-1,3-cyclohexanedione (Mm 328.77; dissoci-ation constant, pKa, 3.13; boiling point, 574.5 °C at 760 mmHg; Riedelde Haen, Pestanal, Saint-Quentin Fallavier, France), and commercialproduct as a concentrated suspension of sulcotrione at 300 g/L (liquidMikado; Bayer CropScience) were used. Water was purified through asterile Millipore Milli-Q system (Millipore αQ; resistivity, 18 M·cm,DOC, <0.1 mg/L).The experimental model of V. faba var. Aguadulce, is a cultivar with
a high sensitivity to pollutants.5,30 V. faba was also used to studyresponses of oxidative stress markers31 and other antitoxic plantdefenses, such as phytochelatins,32 to environmental pollutants. Seedsof V. faba were surface sterilized with 10% sodium hypochloride,rinsed several times with distilled water, and placed on moistenedpaper at 25 °C for 4−5 days, allowing them to germinate, then put inpots filled with commercial substrate (40% black, 30% brown, and 30%blond peat moss, pH 6.1) and transfer to greenhouse (22 ± 5 °C;photoperiod, 16 h light/8 h dark). Grape marc (powder Acys; 60%polyphenols containing 25% anthocyanins) was a product fromGrap’Sud (30360 Cruviers-Lascours).21
Plant Treatments. Herbicides, grape marc, and mixtures wereapplied as aqueous solutions on leaf tissue either as aerosol spray or byinfiltration.Foliar Spray Treatments. Spray treatment (1.12 mL per plant) on
foliage was made onto the upper face of four leaves (two sprays perleaf). Plants were thus treated as in fields, that is, sulcotrione 300 g/haor Mikado, 1 L/ha, a trademark that contains sulcotrione (300 g/L).
Plants were thus treated using 0.3 mg/dm2 (A1) sulcotrione activeingredient (ai) or sulcotrione in commercial product (i.e., 1 μL ofMikado). Treatments with grape marc contained 0.9 mg/dm2 grapeproduct for A1 treatments. The mixture of grape marc and sulcotrionethen had a ratio 3:1 (w/w), respectively. To use similar ratios ofsulcotrione and grape marc for treatments, the mixture made of grapemarc and Mikado was 0.9 mg/1 μL (w/v), respectively. A2 and A3application rates were 2 and 3 times the A1 rate, respectively, for everyproduct. All products and mixtures were solubilized in 6 mL of sterileultrapure water before being sprayed. Control leaf samples weresprayed with 1.12 mL of ultrapure water alone.
Leaf Infiltrations. These were carried out on leaf blades usingplastic syringes. For infiltration treatments, 50 μL of solutions wasusually infiltrated into a leaf area of 1 cm2. Three replicates wereperformed on different plants. Table 1 describes experimentalconditions compared to sulcotrione pesticide used in fields. Fortranscript analysis by leaf infiltrations, C1 treatments used sulcotrioneactive ingredient, Mikado (300 mg/mL), and grape marc (0.9 mg/mL), either alone or in mixtures. C2 and C3 treatments were used 2and 3 times the C1 application rate for each product, respectively.Negative controls were obtained with sterile ultrapure water infiltratedleaves. Plant responses were observed after 2 or 4 days as indicated fortreatments.
Analysis of Membrane Lipoperoxides. Fresh bean roots wereground in a buffer solution consisting of 0.5% thiobarbituric acid(TBA) and 20% trichloroacetic acid (TCA) (w/v = 1:10) with sterileFontainebleau sand. The ground material was heated in a water bath at95 °C for 30 min. During incubation, TBA and aldehyde compoundswere bound. The reaction was stopped by immediate cooling in an icebath. After centrifugation at 10000g for 10 min, the supernatant wasrecovered for a colorimetric assay of lipoperoxides. Their concen-trations were determined according to the method of Heath andPacker.33 The lipid peroxidation products that react with TBA mainlyare malondialdehyde, MDA, and endoperoxides.34 Absorbance of theTBA−MDA complex was measured by spectrophotometer at 532 nm
Table 1. Treatment Protocolsa
pot cultures field
products sulcotrione Mikado grape marc mixtures Mikado
aLeaf spray protocol: plants with four leaves, covering 1 dm2, were sprayed twice on each leaf. Volume of one spray was 0.14 mL. Therefore,treatments for 1 plant (1 dm2) required 1.12 mL of solution, containing 300 μg of sulcotrione or 1 μL of Mikado and/or 900 μg grape marc forapplication rates called A1. Treatments A2 and A3 used 2 and 3 times A1 rates, respectively, for each product. For comparison, the application rate ofsulcotrione used in fields is reported. Leaf inf iltration protocol: usually, 50 μL of liquid (products in ultrapure water) was infiltrated until solutionsspread across leaf area of 1 cm2. C1 treatments used 268 μg/mL sulcotrione solution, either active ingredient or trademark Mikado, and 9 μg/mLgrape marc, either alone or in mixtures. C2 and C3 treatments used 2 and 3 times C1 application rates, respectively, for each product. Forcomparison, the application rate of sulcotrione used in fields is reported.
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and corrected by reading at 600 nm. MDA concentration wascalculated using a molar extinction coefficient (ε = 155 mm/cm).Cell Death Assays. Cell death in leaf tissue was monitored by
Evans Blue staining35 as previously described.21 Each assay wasperformed with 10 leaf disks (1 cm in diameter) punched out fromeach infiltrated area of the same leaf; triplicates were obtained fromthree different plants. Leaf disks were incubated for 30 min in 0.25%Evans Blue (Sigma, France) at room temperature on a shaker,extensively rinsed to remove excess dye, and ground in a tissue grindtube with 1 mL of 1% SDS solution. Treated leaf extracts were thencentrifuged for 20 min at 20000g, and supernatants were diluted 8-foldwith distilled water. Dye absorbencies were measured with aspectrophotometer at 600 nm and referred to the absorbance versusleaf disk weight, for either sprayed or infiltrated plants. Cell death wasmeasured with plants treated by spray, either with differentconcentrations of products (A1−A3), pesticide, and grape marc for4 days, or using the highest rates of products (A3) but for two periods,2 and 4 days.RNA Isolation and Quantification. Leaf tissues (200 mg) were
ground in liquid nitrogen, and RNA extractions were performed using1 mL of Trireagent solution (Euromedex, France) according to themanufacturer’s instructions. Total RNA was cleaned up with 0.5 UDNase I solution (Euromedex, France), containing 20 U RNaseinhibitor (Euromedex, France). RNA integrity was analyzed on 1%agarose gel by observation of rRNA spots. RNA concentrations weremeasured by absorbance at 260 nm. Reverse transcription wasperformed using total RNA and Euroscript Reverse Transcriptase(Eurogentec, France) according to the manufacturer’s instructions.Quantitative assessment of mRNA levels was performed using aniCycler iQv3 (Bio-Rad). PCR reactions were prepared with qPCR kitMastermix for SYBR green (Eurogentec, France) according to themanufacturer’s protocol. The cDNA concentration used thresholdcycle values (CT) between 15 and 30 cycles. Amplification specificitywas checked by melting-curve analysis22 and involved 42 cycles(melting, 95 °C; hybridization, 56 °C; elongation, 30 s for each step).PCR efficiencies were calculated for each gene according to Pfaffl.36
Accumulations of gene transcripts were calculated using referencetranscript and actin gene and set relative to control plants (waterinfiltration). Primer sequences of the studied gene are shown in Table2.Statistical Analysis. All experiments were done in triplicates. Bars
represent the mean of three values ± standard error (SE). Student’stest was used to compare the means for determination of significantdifferences between treated samples and control. The data are written
as the mean ± SE. Values were determined to be significant when p ≤0.05.
■ RESULTS
Cell Death Analyses. Effects of treatments on plantsexposed to the herbicide are illustrated in Figure 1. Sulcotrioneor Mikado sprayed on leaves displayed a significant depletion ofleaf surfaces (Figure 1A) and a higher MDA content in roots(Figure 1B) than control after 2 days of treatment. Treatmentby grape marc alone did not show a significant effect. Asignificant increase was observed in MDA of plant treated bythe mixtures sulcotrione or Mikado and grape marc. Suchperoxidation could be a result of oxidative degradation of lipidsdue to a significant change in the cellular redox status in plantsgenerated by herbicide as previously reported.37 Thisobservation indicated that herbicide spray led to plantmembrane modification.Sulcotrione pesticides, active ingredient and trademark
(Mikado), induced higher cell death rate than observed incontrol leaves. Cell death was also enhanced with trademarkproduct treatments (Figure 2A). Cell death was increased whengrape marc was added either to sulcotrione (ai) or tocommercial product (Figure 2A). Cell death was shown to beconcentration-dependent (Figure 2A). It was triggered by grapemarc or pesticides (sulcotrione or Mikado) treatments, but athigh rates of pesticides. Mixtures then induced a great celldeath, which seems to be related to an addition of producttoxicity for sulcotrione but that still was enhanced with Mikado(Figure 2A).Cell death evolutions were similar for plants treated either
with grape marc or sulcotrione (Figure 2B). Addition of grapemarc to sulcotrione enhanced cell death (Figure 2B). Mikadotreatment displayed quicker cell death than grape marctreatment alone, but a mixture of both products still acceleratedcell death, suggesting a synergy effect (Figure 2B).
Gene Expressions. Accumulations of intracellular ROSbelong to cell death markers. Study of gene expression involvedin oxidative burst metabolism clarified effects of planttreatments. Plants were treated by leaf infiltration with
Table 2. Primer Sequences for Gene Probes
target gene primer sequences product length (bp) accession no.
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application rates called C1 for sulcotrione or Mikado (13.4 μgof active ingredient) and grape marc (40 μg per infiltration).Mixtures of grape marc and sulcotrione products were used at aratio 3:1 (w/w), respectively. C2 and C3 are 2 and 3 times C1rates, respectively, for sulcotrione products or grape marcquantities.Sulcotrione treatments showed an increase of plastid
copper−zinc superoxide dismutase (Cu/Zn-SOD CH) tran-scripts in leaves of V. faba, which was dose-dependent (Figure3A). Other treatments, grape marc or Mikado (Figures 3A and4A), displayed increases of transcripts for high concentrations.Addition of grape marc to herbicide enhanced sulcotrione effect(Figure 3A), but lowered induction by Mikado (Figure 4A).Sulcotrione or Mikado treatments showed increases of
cytosol copper−zinc superoxide dismutase (Cu/Zn-SOD CY)transcripts in leaves of V. faba, which displayed a dose−response relationship (Figures 3B and 4B). These treatmentsenhanced ascorbate peroxidase transcript accumulation in V.faba leaves mainly for the first rate C1, with a slight decrease forother application rates (Figures 3C and 4C). They also showed
increases of catalase, ubiquitin, and hsp 70 transcriptaccumulations in leaves of V. faba that were dose-dependent(Figures 3 and 4D−F, respectively).Grape marc treatments displayed an increase of cytosol
copper−zinc superoxide dismutase (Cu/Zn-SOD CY) tran-scripts; addition of grape marc to herbicide enhanced effects ofsulcotrione and Mikado for all rates (Figures 3B and 4B). Incontrast, grape marc treatments displayed ascorbate peroxidasetranscript increases that are dose-dependent (Figures 3C and4C); addition of grape marc to herbicide did not markedlychange effects of sulcotrione and Mikado (Figures 3C and 4C).Grape marc treatments displayed an increase of catalasetranscript accumulation from the lowest rate C1 (Figures 3Dand 4D); addition of grape marc to herbicide enhanced effectsof sulcotrione and Mikado, at least for C1and C2 applicationrates; in contrast, the C3 rate lowered Mikado induction,whereas the effect of sulcotrione still was enhanced (Figures 3Dand 4D). Addition of grape marc to herbicide also enhancedubiquitin and hsp 70 transcript accumulations by sulcotrioneand Mikado for every rate (Figures 3E,F and 4E,F,respectively).
Figure 1. Physiological changes of plant, 2 days after treatments, byspray at application rate A3 for each product, as described in Table 1:(A) surfaces of treated V. faba leaves; (B) malondialdehyde content inV. faba roots. Control leaves were sprayed with ultrapure water. Eachindependent experiment was performed in triplicates. Bars representmean values ± SE. Stars are significant differences between treatmentsamples and control, and a and b letters are significant differencesbetween mixture treatments and corresponding herbicide alone (p <0.05).
Figure 2. Cell death analysis. (A) Cell death induced after 4 days oftreatments of V. faba leaves by spray. Treatments were done withsulcotrione, Mikado, grape marc, and a mixture of components,compared with V. faba control leaves. Application rates for planttreatment (A1) were 0.3 mg/dm2 sulcotrione (ai) or in commercialproduct (i.e., 1 μL of Mikado). Grape marc was used at a rate 0.9 mg/dm2 for A1. Mixture of grape marc and sulcotrione was used at a ratio3:1 (w/w), respectively. Mixture made of grape marc and Mikado hada ratio 0.9:1 (w/v), respectively, to obtain a similar ratio forsulcotrione quantities. A2 and A3 were 2 and 3 times A1 rates foreach product, respectively. Control samples were sprayed out withultrapure water. Dye absorbance was measured at 600 nm. (B) Celldeath induced by different spray treatment in leaves. Beans weretreated by the highest product concentrations (A3). Absorbencieswere measured after 2 and 4 days of treatments.
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■ DISCUSSION
We have shown that herbicide can trigger oxidative stressresulting in disruption of cell metabolism, in experimentalconditions that did not display leaf bleaching. Sulcotrione, asactive ingredient or in a formulated product, Mikado, inducedaccumulation of MDA; it was suggested that pure sulcotrioneor Mikado may induce cell membrane destruction, as waspreviously reported.38 Lipid peroxidation has been suggested asone of the mechanisms involved in toxicity induced bypesticides.39 Treatments of V. faba leaves with herbicidessulcotrione or Mikado displayed cell death increases thatchanged according to active ingredient concentrations and wereenhanced using trademark Mikado. Grape marc also displayedslight bean cell death in a dose-dependent manner. Addition ofgrape marc either to sulcotrione or Mikado enhanced celldeath, with higher increases using Mikado than with activeingredient alone. A potentiating effect between grape marcaction and adjuvant elements of Mikado was then suggested.
Commercial product Mikado showed greater cell toxicitythan active product sulcotrione. This was previously shownwith mesotrione and Callisto or glyphosate and Roundup usinggrowth inhibition criteria in several toxicity test organisms.28,29
Mikado was shown to be toxic for Pseudokirchneriellasubcapitata growth, whereas its toxicity was quite reduced forChlorella vulgaris.2 Pesticide action thus may strongly enhanceecotoxicological effects of other chemicals at high mixtureconcentrations, thereby showing potential synergy. Adjuvantadded to herbicide formulations could enhance cell toxicity, aswas potentially the case for methanol in Viper.40 In contrast, itwas previously shown that absorption of sulcotrione in maizewas not significantly enhanced by adjuvant contained incommercial Mikado.41 Moreover, most of the absorbedsulcotrione remained in the treated leaf and <14% wastranslocated to other organs; the presence of adjuvant inMikado did not significantly modify the translocation ofsulcotrione.41
Figure 3. Transcript accumulation in V. faba leaves after infiltration treatments with sulcotrione, grape marc, and mixture of both components: (A)chloroplast copper−zinc superoxide (Cu/Zn-SOD CH) dismutase; (B) cytosolic copper−zinc superoxide dismutase (Cu/Zn-SOD CY); (C)ascorbate peroxidase (APX); (D) catalase; (E) ubiquitinl; (F) heat shock protein hsp 70.1. Values of treated leaves, with both sulcotrione or Mikadoand grape marc, that have letters “a” or “b” are statistically different from values of treated leaves by sulcotrione or Mikado alone, at 5 or 1%significance level, respectively, according to Student’s t test. Values with one or two stars are statistically different from control at 5 and 1%significance level, respectively. C2 and C3 treatments used 2 and 3 times, respectively, C1 rates for each product as described in Table 1.
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Earlier, it was observed that formulation surprisinglyincreased the rate of sulcotrione phototransformation.12
Synergistic joint effects on survival of daphnids for fungicide/insecticide and fungicide/herbicide mixtures42 and for mixturescomposed with only herbicides were found.2,43 It was oftenexplained that the effect of one chemical could be synergized bythe presence of others, usually regulated by changes ofdetoxifying/biotransformation processes.42 Recently, we haveshown that sulcotrione and photoproducts increase chromoso-mal abnormality frequencies, but photodegraded sulcotrionecocktail has shown a greater toxicity than the parentmolecule.11
Polyphenols can directly influence different points ofapoptotic process and/or expression of regulatory proteins.44
Mosse et al.45 have shown that winery wastewater delayedgermination and inhibited vegetative growth of several plants.We have studied and shown variations of oxidative stress inbean leaves exposed to pesticides and grape marc, measuringtranscript accumulations of stress gene markers, such assuperoxide dismutases, catalase, ascorbate peroxidase, ubiquitin,and hsp 70, as previously described.46−50 Agati et al.51 recentlyproposed a model in which chloroplast-located flavonoids
scavenge generated ROS, thus avoiding programmed cell death.They suggested that flavonoids in conjunction with peroxidasesand ascorbic acid constitute a secondary antioxidant system. Incontrast, we showed that the addition of grape marc toherbicide products, sulcotrione or Mikado, had various effectsaccording to herbicide composition (active ingredient alone orcommercial product). Marker genes are differently expressed.Mixtures of grape marc and herbicide enhanced transcriptaccumulation for ubiquitin, hsp 70, and cytosolic SOD genes.Mixtures of grape marc and sulcotrione or Mikado gavedifferent results at the highest rates for chloroplast SOD andcatalase transcript accumulations. Grape marc mixturesmarkedly did not change effects of sulcotrione and Mikadofor ascorbate peroxidase transcript accumulation. Depletion ofcatalase activity was previously reported in fishes exposed toendosulfan (ES) and was attributed to flux of superoxideradicals.52,53
Chromosome breakage, misrepaired DNA lesions, orchromosome unsegregation can be induced by oxidativestress54,55 and could explain a genotoxic effect of sulcotrionepreviously described.5 Other herbicide and pesticide chemicalssuch as insecticides may induce oxidative stress leading to
Figure 4. Transcript accumulation in V. faba leaves after infiltration treatments with Mikado, grape marc, and mixture of both components: (A)chloroplast copper−zinc superoxide (Cu/Zn-SOD CH) dismutase; (B) cytosolic copper−zinc superoxide dismutase (Cu/Zn-SOD CY); (C)ascorbate peroxidase (APX); (D) catalase; (E) ubiquitin; (F) heat shock protein hsp 70.1. Treatment values with letters “a” or “b” are statisticallydifferent from pesticide treatment at 5 or 1% significance level, respectively, and those with one or two stars are statistically different from control at5 and 1% significance level, respectively, according to Student’s t test. C2 and C3 treatments used 2 and 3 times, respectively, C1 rates for eachproduct as described in Table 1.
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generation of free radicals and alterations in antioxidative orfree radical scavenging enzyme systems.56−59 It was indicatedthat enzymes associated with antioxidative defense mechanismsare altered under the influence of pesticides.59 Moreover,oxidative stress and DNA damage have been proposed asmechanisms linking pesticide exposure to health effects such ascancer and neurological diseases.60
Proteins that are involved in cell stress metabolism, such ashsp 70 and ubiquitin, showed transcript increase followingtreatments by either sulcotrione or Mikado on V. faba leaves.Grape marc treatments alone displayed an increased accumu-lation of the same transcripts. In our conditions, mixtures ofgrape marc and herbicide enhanced effects of sulcotrione orMikado for each concentration.Earlier, it has been shown that intracellular ROS accumu-
lation was attenuated by pretreatment with procyanidin B2.61
In addition to antioxidative capacity, polyphenols may exert aprotective effect by selective inhibition or stimulation of keyprotein into cell signaling cascades. Flavanols are also potentantioxidants in lipid systems, where they reduce oxidativemodifications of membranes by restricting access of oxidants tothe bilayer and propagation of lipid oxidation in hydrophobicmembrane matrix.62 In different experimental conditions,changes in catalase (CAT) and superoxide dismutase (SOD)gene expressions were observed after chronic consumption ofwine phenolics by men.63
In conclusion, transcript accumulations of described geneswere shown to be enhanced after treatments of leaves at threedifferent product concentrations of sulcotrione or Mikado,grape marc, and mixtures. Genes that are involved in oxidativespecies induction (cytoplasmic and plastid SODs),64 thatencode antioxidative enzymes (catalase and ascorbate perox-idase, APX)65 or which are involved in cellular defense (hsp 70and ubiquitin)66−69 were up-regulated after leaf treatments bypesticides and grape marc. Finally, cell death was increased aftertreatments but with high rates for pesticide cocktailssupplemented with grape marc, especially for Mikado (Figure5). Expression of these genes was also involved in theregulation of different cellular signalization pathways eithertriggering (SOD) or inhibiting various signal ways (Figure 5).Using suspension-cultured cells of tobacco, grape marc wasshown to elicit early perception events and, then activatingdefense-related genes, ult imately leading to cel ldeath.70Pesticide, sulcotrione or Mikado, and grape marc,alone, can induce target plant cell death. Signal transductionmay involve ROS as shown in plant stressed conditions.64,71 Amixture of pesticide and grape marc suggests a potentiatingeffect of grape marc by adjuvant elements of Mikado.Sulcotrione and mesotrione are herbicides related to a naturalβ-triketone, leptospermone from Leptospermum scoparium, thatspecifically inhibits the enzyme HPPD. Plant β-triketones alsopossess an array of biological activities such as antimicrobialactivities and may protect plants from biotic stresses.72
Leptospermone has shown toxicity against mite development,a nonherbicide target.73
Sulcotrione doses used for gene expression studies weresimilar for spray treatments or 3 to 4 times higher forinfiltration treatments than the sulcotrione amount used inagriculture, as shown in Table 1. Herbicides and grape marc cantrigger oxidative stress, resulting in disruption of cellularmetabolism. We provide evidence that mixtures (herbicide andgrape marc) induced specific gene expression for stress-associated proteins. Addition of grape marc to herbicide
products, sulcotrione or Mikado, had different effects accordingto herbicide composition (active product or commercialproduct) and, meanwhile, genes are shown to be differentlyexpressed. Therefore, adjuvant can modify the biologicalfeatures of sulcotrione. Moreover, grape marc can enhancethe biological effects of sulcotrione and Mikado but withdifferent gene expression features.
■ AUTHOR INFORMATIONCorresponding Author*(G.L.) Phone: +33 473407908. Fax: +33 473407951. E-mail:[email protected] work was supported by grants from Europe (FEDER) andRegion Auvergne Council.NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSWe thank Celine Sac and Dominique Marcon for their skillfulassistance.
■ REFERENCES(1) Dayan, F. E.; Cantrell, C. L.; Duke, S. O. Natural products in cropprotection. Bioorg. Med. Chem. 2009, 17, 4022−4034.(2) Marques, C. R.; Goncalves, A. M. M.; Pereira, R.; Goncalves, F.Ecotoxicological effects of Mikado® and Viper® on algae anddaphnids. Environ. Toxicol. 2012, 27, 685−699.(3) Wiszniowski, J.; ter Halle, A.; Richard, C.; Hitmi, A.; Ledoigt, G.Photodegradation product of sulcotrione and the physiologicalresponse of maize (Zea mays) and white mustard (Sinapis alba).Chemosphere 2009, 74, 1224−1230.(4) Chaabane, H.; Vulliet, E.; Joux, F.; Lantoine, F.; Conan, P.;Cooper, J. F.; Coste, C. M. Photodegradation of sulcotrione in various
Figure 5. Putative interactions of products, such as herbicide(sulcotrione or Mikado) or grape marc, with cellular signalizationpathways that can trigger cell defenses or death. Gene expressions areshown activating (arrow) or inhibiting (dash lines) signalizationpathways. Gene markers are involved in accumulation of oxidativespecies (cytoplasmic and plastid superoxide dismutases, SOD, catalase,and ascorbate peroxidase, APX) or in cell defense (hsp 70 andubiquitin). Cell death is shown to be increased after treatments usinghigh application rates when pesticide cocktails with grape marc wereused, especially associated with Mikado, as displayed by arrowthickness.
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