Article pubs.acs.org/JAFC
Toxicity of Sulcotrione and Grape Marc on Vicia faba Cells Chaima Sta,†,‡,§ Eric Goujon,†,‡ Ezzeddine Ferjani,§ and Gérard Ledoigt*,†,‡ †
Clermont Université, Université Blaise Pascal, UMR 547 PIAF, B.P. 10448, F-63000 Clermont-Ferrand, France Campus Universitaire des Cézeaux, 24 Avenue des Landais, 63177 Aubière cedex, France § Laboratoire de Physiologie et Génétique des Plantes à Intérêt Agronomique, Faculté des Sciences de Bizerte, Université de Carthage, 7021 Jarzouna, Tunisia ‡
ABSTRACT: The cell toxicity of sulcotrione, a selective triketone herbicide, was evaluated on Vicia faba. Sulcotrione, trademark Mikado, grape marc, and mixtures of sulcotrione or Mikado with grape marc induced cell death. Addition of grape marc to either sulcotrione or Mikado enhanced cell death, especially with Mikado. Addition of grape marc to herbicides, sulcotrione, or Mikado resulted in different expression of genes usually associated with cell stress. Mixtures of grape marc and herbicides enhanced transcript accumulation for ubiquitin, hsp 70, and cytosolic superoxide dismutase, but did not change ascorbate peroxidase transcript accumulation. The results thus provide evidence that sulcotrione, Mikado, and mixtures with grape marc can trigger cell death and specific gene expressions. Cocktails of products with sulcotrione, such as commercial additives and grape marc, can modify biological features of pesticide. Moreover, grape marc differently enhanced cell toxicity of sulcotrione and Mikado, suggesting a synergy between pesticide products and grape marc. KEYWORDS: cell death, gene expression, grape marc, sulcotrione, Vicia faba
■
INTRODUCTION Conventional pest management has been significantly influenced by bioactive natural products.1 Mikado (Bayer Crop Science) is a systemic foliar-applied postemergence herbicide mostly used for corn crops to control broadleaf weeds, 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 1−2 L/ha in fields. In culture, maize population density was 100,000 plants per hectare, and application rate amounts were up to 300 g of sulcotrione/ha, which yields 3 mg of active substance per plant (or 30 mg/m2).3 Triketone herbicides inhibit the chain of photosynthetic electron transfer, therefore blocking a mechanism of energy production in plant.3−5 Sulcotrione vapor pressure value, 5 × 10−6 Pa at 25 °C, indicates a low volatile compound. In an aqueous medium at 25 °C, sulcotrione was shown chemically stable with a half-life estimated to be between 200 and 400 days depending on the pH.3 EFSA6 reported that the half-life for the degradation of sulcotrione ranged from 6 to 15 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 is mobile, with a Koc of 36 L/kg, and will not tend to adsorb to suspended solids and sediment.6,7 Sulcotrione, 2-[2-chloro-(4-methylsulfonyl)benzoyl]-1,3-cyclohexanedione, can be absorbed by leaves and by root system and may accumulate in the soil more than a month after application. Water solubility of the product is 165 mg/L at 25 °C with a great potential to leach.8−10 It belongs to the triketone herbicides that inhibit 4-hydroxyphenylpyruvate dioxygenase enzyme (p-HPPD), leading to carotenoid rate decrease in weeds. Loss of carotenoid and α-tocopherol can result in photosynthesis inhibition, triggering accumulation of reactive oxygen species (ROS).2,4 In another biological © XXXX American Chemical Society
pathway, sulcotrione induced chromosomal alterations, indicating potent mutagen effects.5,11 Recent studies have shown that exposure to sunlight can be one of the most destructive factors for pesticides following crop treatment that can generate toxic byproducts4,12,13 in the environment; it can play a significant role for pesticide effects on both human health and natural ecosystems.14−17 Grape marc has been patented as a new class of photoprotecting agent18 that allows pesticide photodegradation to be reduced.19,20 It was shown to trigger an array of plant defense responses, making this natural compound a potential phytosanitary product with an issue for sustainable agriculture and environmentally friendly practices.21 Grape marc is an anthocyanin-rich mixture typically containing about 60% of polyphenols, among which 8−25% are anthocyanins, and was obtained from grape pomace.21,22 Anthocyans belong to the large family of flavonoids extensively widespread in plants and display antioxidative capacity.23 Association between anthocyanins and oxidative stress can result in anthocyanin ability to increase photoprotection and, hence, to reduce a putative oxidative damage.24,25 Vicia faba is a dicotyledonous plant species that showed a great susceptibility to clastogenic and cytotoxic chemicals26,27 and belongs to a plant family susceptible to Mikado according to Bayer Crop Science. It was therefore used as a target plant model for sulcotrione treatments. Earlier papers described different effects on plants using active molecule or the trademark product.28,29 Therefore, we have compared treatments using either the active ingredient sulcotrione or the Received: July 14, 2014 Revised: October 8, 2014 Accepted: October 21, 2014
A
dx.doi.org/10.1021/jf503323t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 1. Treatment Protocolsa field
pot cultures products
sulcotrione
Mikado
grape marc Spray 1.12 mL
liquid volumes area (four leaves) application rates A1 300 μg
1 μL (300 μg sulcotrione)
900 μg
A2
600 μg
2 μL (600 μg sulcotrione)
1800 μg
A3
900 μg
3 μL (900 μg sulcotrione)
2700 μg
liquid volumes area application rates C1 300 μg/1.12 mL, i.e., 13.4 μg/50 μL/cm2 C2 27 μg/50 μL/cm2 C3
40 μg/50 μL/cm2
mixtures
Mikado 1L
1 dm2
1 ha
300 μg sulcotrione/900 μg grape marc/ dm2 600 μg sulcotrione/1800 μg grape marc /dm2 900 μg sulcotrione/2700 μg grape marc/ dm2
300 g sulcotrione/ha, i.e., 300 μg/dm2
Infiltration 50 μL
1L
1 cm2
1 ha
1 μL (300 μg sulcotrione)/1.12 mL, i.e., 13.4 μg sulcotrione/50 μL/cm2 27 μg sulcotrione/50 μL/cm2
900 μg/1.12 mL, i.e., 40 μg/50 μL/cm−2 80 μg/50 μL/cm−2
40 μg sulcotrione/50 μL/cm2
120 μg grape marc/ 50 μL/cm2
13.4 μg sulcotrione/40 μg grape marc/ 50 μL/cm2 27 μg sulcotrione/80 μg grape marc/ 50 μL/cm2 40 μg sulcotrione/120 μg grape marc/ 50 μL/cm2
300 g/ha, i.e., 3 μg/cm2
a
Leaf 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 for application rates called A1. Treatments A2 and A3 used 2 and 3 times A1 rates, respectively, for each product. For comparison, the application rate of sulcotrione used in fields is reported. Leaf inf iltration protocol: usually, 50 μL of liquid (products in ultrapure water) was infiltrated until solutions spread across leaf area of 1 cm2. C1 treatments used 268 μg/mL sulcotrione solution, either active ingredient or trademark Mikado, and 9 μg/mL grape marc, either alone or in mixtures. C2 and C3 treatments used 2 and 3 times C1 application rates, respectively, for each product. For comparison, the application rate of sulcotrione used in fields is reported. Plants were thus treated using 0.3 mg/dm2 (A1) sulcotrione active ingredient (ai) or sulcotrione in commercial product (i.e., 1 μL of Mikado). Treatments with grape marc contained 0.9 mg/dm2 grape product for A1 treatments. The mixture of grape marc and sulcotrione then had a ratio 3:1 (w/w), respectively. To use similar ratios of sulcotrione and grape marc for treatments, the mixture made of grape marc and Mikado was 0.9 mg/1 μL (w/v), respectively. A2 and A3 application rates were 2 and 3 times the A1 rate, respectively, for every product. All products and mixtures were solubilized in 6 mL of sterile ultrapure water before being sprayed. Control leaf samples were sprayed with 1.12 mL of ultrapure water alone. Leaf Infiltrations. These were carried out on leaf blades using plastic syringes. For infiltration treatments, 50 μL of solutions was usually infiltrated into a leaf area of 1 cm2. Three replicates were performed on different plants. Table 1 describes experimental conditions compared to sulcotrione pesticide used in fields. For transcript analysis by leaf infiltrations, C1 treatments used sulcotrione active ingredient, Mikado (300 mg/mL), and grape marc (0.9 mg/ mL), either alone or in mixtures. C2 and C3 treatments were used 2 and 3 times the C1 application rate for each product, respectively. Negative controls were obtained with sterile ultrapure water infiltrated leaves. Plant responses were observed after 2 or 4 days as indicated for treatments. Analysis of Membrane Lipoperoxides. Fresh bean roots were ground in a buffer solution consisting of 0.5% thiobarbituric acid (TBA) and 20% trichloroacetic acid (TCA) (w/v = 1:10) with sterile Fontainebleau sand. The ground material was heated in a water bath at 95 °C for 30 min. During incubation, TBA and aldehyde compounds were bound. The reaction was stopped by immediate cooling in an ice bath. After centrifugation at 10000g for 10 min, the supernatant was recovered for a colorimetric assay of lipoperoxides. Their concentrations were determined according to the method of Heath and Packer.33 The lipid peroxidation products that react with TBA mainly are malondialdehyde, MDA, and endoperoxides.34 Absorbance of the TBA−MDA complex was measured by spectrophotometer at 532 nm
commercial product Mikado. The aim of the work was to evaluate consequences of treatments by sulcotrione and trademark Mikado on plant cell physiology and the ability of grape marc to control plant response to determine if such treatments can modify pesticide effects on stress metabolism.
■
MATERIALS AND METHODS
Plant Material and Chemicals. Sulcotrione, 2-(2-chloro-4(methylsulfonyl) benzoyl)-1,3-cyclohexanedione (Mm 328.77; dissociation constant, pKa, 3.13; boiling point, 574.5 °C at 760 mmHg; Riedel de Haën, Pestanal, Saint-Quentin Fallavier, France), and commercial product as a concentrated suspension of sulcotrione at 300 g/L (liquid Mikado; Bayer CropScience) were used. Water was purified through a sterile Millipore Milli-Q system (Millipore αQ; resistivity, 18 M·cm, DOC,
Toxicity of Sulcotrione and Grape Marc on Vicia faba Cells Chaima Sta,†,‡,§ Eric Goujon,†,‡ Ezzeddine Ferjani,§ and Gérard Ledoigt*,†,‡ †
Clermont Université, Université Blaise Pascal, UMR 547 PIAF, B.P. 10448, F-63000 Clermont-Ferrand, France Campus Universitaire des Cézeaux, 24 Avenue des Landais, 63177 Aubière cedex, France § Laboratoire de Physiologie et Génétique des Plantes à Intérêt Agronomique, Faculté des Sciences de Bizerte, Université de Carthage, 7021 Jarzouna, Tunisia ‡
ABSTRACT: The cell toxicity of sulcotrione, a selective triketone herbicide, was evaluated on Vicia faba. Sulcotrione, trademark Mikado, grape marc, and mixtures of sulcotrione or Mikado with grape marc induced cell death. Addition of grape marc to either sulcotrione or Mikado enhanced cell death, especially with Mikado. Addition of grape marc to herbicides, sulcotrione, or Mikado resulted in different expression of genes usually associated with cell stress. Mixtures of grape marc and herbicides enhanced transcript accumulation for ubiquitin, hsp 70, and cytosolic superoxide dismutase, but did not change ascorbate peroxidase transcript accumulation. The results thus provide evidence that sulcotrione, Mikado, and mixtures with grape marc can trigger cell death and specific gene expressions. Cocktails of products with sulcotrione, such as commercial additives and grape marc, can modify biological features of pesticide. Moreover, grape marc differently enhanced cell toxicity of sulcotrione and Mikado, suggesting a synergy between pesticide products and grape marc. KEYWORDS: cell death, gene expression, grape marc, sulcotrione, Vicia faba
■
INTRODUCTION Conventional pest management has been significantly influenced by bioactive natural products.1 Mikado (Bayer Crop Science) is a systemic foliar-applied postemergence herbicide mostly used for corn crops to control broadleaf weeds, 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 1−2 L/ha in fields. In culture, maize population density was 100,000 plants per hectare, and application rate amounts were up to 300 g of sulcotrione/ha, which yields 3 mg of active substance per plant (or 30 mg/m2).3 Triketone herbicides inhibit the chain of photosynthetic electron transfer, therefore blocking a mechanism of energy production in plant.3−5 Sulcotrione vapor pressure value, 5 × 10−6 Pa at 25 °C, indicates a low volatile compound. In an aqueous medium at 25 °C, sulcotrione was shown chemically stable with a half-life estimated to be between 200 and 400 days depending on the pH.3 EFSA6 reported that the half-life for the degradation of sulcotrione ranged from 6 to 15 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 is mobile, with a Koc of 36 L/kg, and will not tend to adsorb to suspended solids and sediment.6,7 Sulcotrione, 2-[2-chloro-(4-methylsulfonyl)benzoyl]-1,3-cyclohexanedione, can be absorbed by leaves and by root system and may accumulate in the soil more than a month after application. Water solubility of the product is 165 mg/L at 25 °C with a great potential to leach.8−10 It belongs to the triketone herbicides that inhibit 4-hydroxyphenylpyruvate dioxygenase enzyme (p-HPPD), leading to carotenoid rate decrease in weeds. Loss of carotenoid and α-tocopherol can result in photosynthesis inhibition, triggering accumulation of reactive oxygen species (ROS).2,4 In another biological © XXXX American Chemical Society
pathway, sulcotrione induced chromosomal alterations, indicating potent mutagen effects.5,11 Recent studies have shown that exposure to sunlight can be one of the most destructive factors for pesticides following crop treatment that can generate toxic byproducts4,12,13 in the environment; it can play a significant role for pesticide effects on both human health and natural ecosystems.14−17 Grape marc has been patented as a new class of photoprotecting agent18 that allows pesticide photodegradation to be reduced.19,20 It was shown to trigger an array of plant defense responses, making this natural compound a potential phytosanitary product with an issue for sustainable agriculture and environmentally friendly practices.21 Grape marc is an anthocyanin-rich mixture typically containing about 60% of polyphenols, among which 8−25% are anthocyanins, and was obtained from grape pomace.21,22 Anthocyans belong to the large family of flavonoids extensively widespread in plants and display antioxidative capacity.23 Association between anthocyanins and oxidative stress can result in anthocyanin ability to increase photoprotection and, hence, to reduce a putative oxidative damage.24,25 Vicia faba is a dicotyledonous plant species that showed a great susceptibility to clastogenic and cytotoxic chemicals26,27 and belongs to a plant family susceptible to Mikado according to Bayer Crop Science. It was therefore used as a target plant model for sulcotrione treatments. Earlier papers described different effects on plants using active molecule or the trademark product.28,29 Therefore, we have compared treatments using either the active ingredient sulcotrione or the Received: July 14, 2014 Revised: October 8, 2014 Accepted: October 21, 2014
A
dx.doi.org/10.1021/jf503323t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 1. Treatment Protocolsa field
pot cultures products
sulcotrione
Mikado
grape marc Spray 1.12 mL
liquid volumes area (four leaves) application rates A1 300 μg
1 μL (300 μg sulcotrione)
900 μg
A2
600 μg
2 μL (600 μg sulcotrione)
1800 μg
A3
900 μg
3 μL (900 μg sulcotrione)
2700 μg
liquid volumes area application rates C1 300 μg/1.12 mL, i.e., 13.4 μg/50 μL/cm2 C2 27 μg/50 μL/cm2 C3
40 μg/50 μL/cm2
mixtures
Mikado 1L
1 dm2
1 ha
300 μg sulcotrione/900 μg grape marc/ dm2 600 μg sulcotrione/1800 μg grape marc /dm2 900 μg sulcotrione/2700 μg grape marc/ dm2
300 g sulcotrione/ha, i.e., 300 μg/dm2
Infiltration 50 μL
1L
1 cm2
1 ha
1 μL (300 μg sulcotrione)/1.12 mL, i.e., 13.4 μg sulcotrione/50 μL/cm2 27 μg sulcotrione/50 μL/cm2
900 μg/1.12 mL, i.e., 40 μg/50 μL/cm−2 80 μg/50 μL/cm−2
40 μg sulcotrione/50 μL/cm2
120 μg grape marc/ 50 μL/cm2
13.4 μg sulcotrione/40 μg grape marc/ 50 μL/cm2 27 μg sulcotrione/80 μg grape marc/ 50 μL/cm2 40 μg sulcotrione/120 μg grape marc/ 50 μL/cm2
300 g/ha, i.e., 3 μg/cm2
a
Leaf 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 for application rates called A1. Treatments A2 and A3 used 2 and 3 times A1 rates, respectively, for each product. For comparison, the application rate of sulcotrione used in fields is reported. Leaf inf iltration protocol: usually, 50 μL of liquid (products in ultrapure water) was infiltrated until solutions spread across leaf area of 1 cm2. C1 treatments used 268 μg/mL sulcotrione solution, either active ingredient or trademark Mikado, and 9 μg/mL grape marc, either alone or in mixtures. C2 and C3 treatments used 2 and 3 times C1 application rates, respectively, for each product. For comparison, the application rate of sulcotrione used in fields is reported. Plants were thus treated using 0.3 mg/dm2 (A1) sulcotrione active ingredient (ai) or sulcotrione in commercial product (i.e., 1 μL of Mikado). Treatments with grape marc contained 0.9 mg/dm2 grape product for A1 treatments. The mixture of grape marc and sulcotrione then had a ratio 3:1 (w/w), respectively. To use similar ratios of sulcotrione and grape marc for treatments, the mixture made of grape marc and Mikado was 0.9 mg/1 μL (w/v), respectively. A2 and A3 application rates were 2 and 3 times the A1 rate, respectively, for every product. All products and mixtures were solubilized in 6 mL of sterile ultrapure water before being sprayed. Control leaf samples were sprayed with 1.12 mL of ultrapure water alone. Leaf Infiltrations. These were carried out on leaf blades using plastic syringes. For infiltration treatments, 50 μL of solutions was usually infiltrated into a leaf area of 1 cm2. Three replicates were performed on different plants. Table 1 describes experimental conditions compared to sulcotrione pesticide used in fields. For transcript analysis by leaf infiltrations, C1 treatments used sulcotrione active ingredient, Mikado (300 mg/mL), and grape marc (0.9 mg/ mL), either alone or in mixtures. C2 and C3 treatments were used 2 and 3 times the C1 application rate for each product, respectively. Negative controls were obtained with sterile ultrapure water infiltrated leaves. Plant responses were observed after 2 or 4 days as indicated for treatments. Analysis of Membrane Lipoperoxides. Fresh bean roots were ground in a buffer solution consisting of 0.5% thiobarbituric acid (TBA) and 20% trichloroacetic acid (TCA) (w/v = 1:10) with sterile Fontainebleau sand. The ground material was heated in a water bath at 95 °C for 30 min. During incubation, TBA and aldehyde compounds were bound. The reaction was stopped by immediate cooling in an ice bath. After centrifugation at 10000g for 10 min, the supernatant was recovered for a colorimetric assay of lipoperoxides. Their concentrations were determined according to the method of Heath and Packer.33 The lipid peroxidation products that react with TBA mainly are malondialdehyde, MDA, and endoperoxides.34 Absorbance of the TBA−MDA complex was measured by spectrophotometer at 532 nm
commercial product Mikado. The aim of the work was to evaluate consequences of treatments by sulcotrione and trademark Mikado on plant cell physiology and the ability of grape marc to control plant response to determine if such treatments can modify pesticide effects on stress metabolism.
■
MATERIALS AND METHODS
Plant Material and Chemicals. Sulcotrione, 2-(2-chloro-4(methylsulfonyl) benzoyl)-1,3-cyclohexanedione (Mm 328.77; dissociation constant, pKa, 3.13; boiling point, 574.5 °C at 760 mmHg; Riedel de Haën, Pestanal, Saint-Quentin Fallavier, France), and commercial product as a concentrated suspension of sulcotrione at 300 g/L (liquid Mikado; Bayer CropScience) were used. Water was purified through a sterile Millipore Milli-Q system (Millipore αQ; resistivity, 18 M·cm, DOC,
