Jasmonic Acids Facilitate the Degradation and Detoxification of Herbicide Isoproturon Residues in Wheat Crops (Triticum aestivum)

Li Ya Ma, Shu Hao Zhang, Jing Jing Zhang,§ Ai Ping Zhang, Na Li, Xin Qiang Wang, Qian Qian Yu, and Hong Yang
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China
State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, China ; College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China

[ABSTRACT]
Jasmonic acid (JA) [or methyl-jasmonic acid (MeJA)] is one of the important regulators of plant growth, development, and defense with respect to environmental stresses, but how JA is involved in mediation of pesticide accumulation and degradation in plants is largely unknown. This study investigated the contribution of MeJA to detoxification and degradation of isoproturon (IPU) residues in wheat (Triticum aestivum). Wheat plants were exposed to 4 mg of isoproturon kg−1 (environmentally realistic concentration). The level of growth and chlorophyll concentration were reduced, while the electrolyte permeability in plants was enhanced. When plants were sprayed with 0.1 μM MeJA, the phytotoxicity induced by isoproturon was significantly assuaged, which was manifested by an increased chlorophyll concentration and a reduced level of cellular damage in wheat. Activities of several stress marker enzymes with isoproturon were repressed in the presence of MeJA. We measured accumulation of isoproturon in wheat and its residues in soil by highperformance liquid chromatography. The concentration of isoproturon in wheat and soils with MeJA was drastically reduced. Using ultraperformance liquid chromatography−tandem mass spectrometry, 12 isoproturon derivatives (eight metabolites and four conjugates) in wheat were characterized. We further provided evidence that the concentration of endogenous MeJA was significantly increased in IPU-exposed plants. These results suggest that MeJA was able to detoxify or degrade isoproturon in wheat when grown in a realistic environmental isoproturon-polluted soil.

[Determination of Endogenous MeJA in Plants]
The assay of internal MeJM was conducted using the specific methyl jasmonate (meja) enzyme-linked immunoassay (ELISA) kit with the double antibody sandwich method (http://www.jonln.com/) [1]. Briefly, the MeJA extract from plants captures the antibody and encapsulates the antibody onto the micropore plate to make the solid phase antibody. Then, the sample MeJA was added to the encapsulated micropore and combined with the labeled antibody to form the antibody antigenenzyme-labeled antibody complex. After a thorough washing, the substrate TMB was added and colored. The color is positively correlated with the content of MeJA. The absorbance was determined at 450 nm, and the content of MeJA was calculated with a standard curve.

Figure 1. Effect of MeJA on TBARS concentrations and IPU on MeJA concentrations in wheat. (A) One-week-old wheat growing in the soil with 4 mg of isoproturon kg−1 was treated with MeJA (0, 0.1, 1, 10, and 100 μM MeJA spray) for 6 days. (B) One-week-old wheat growing in a 0, 0.25, 0.5, 1, and 2 mg L−1 nutrient solution for 72 h. Roots and shoots of plants were separately harvested and analyzed. Values are means ± standard deviations (n = 3). Data denoted with different letters are significantly different (p < 0.05).

Figure 2. Effects of IPU and/or MeJA on (A) elongation, (B) dry mass, (C) electrolyte content, and (D) chlorophyll content of wheat. Seedlings grew in soils with IPU (4 mg kg−1) and without IPU (CK, control) for 4 days. After that, the leaves were sprayed with 0.1 μmol L−1 MeJA once a day for 6 days. Values are means ± standard deviations (n = 3). Data denoted with different letters are significantly different (p < 0.05).

Figure 3. Accumulation of IPU in shoots and roots of wheat (A) and rhizosphere soil (RS), bulk soil (BS, whole soil including rhizosphere and nonrhizosphere), and nonrhizosphere soil (NRS) (B) in different treatments. Seedlings grew in soils with IPU (4 mg kg−1) and without IPU (CK, control) for 4 days. After that, the leaves were sprayed with 0.1 μmol L−1 MeJA once a day for 6 days. Values are means ± standard deviations (n = 3). Data denoted with different letters are significantly different (p < 0.05).

Figure 4. Effect of IPU and/or MeJA on (A) catalase (CAT), (B) peroxidase (POD), (C) ascorbate peroxidase (APX), (D) glutathione Stransferase (GST), and (E) polyphenol oxidase (PPO) activities in shoots and roots of wheat. Seedlings grew in soils with IPU (4 mg kg−1) and without IPU (CK, control) for 4 days. After that, the leaves were sprayed with 0.1 μmol L−1 MeJA once a day for 6 days. Values are means ± standard deviations (n = 3). Data denoted with different letters are significantly different (p < 0.05).

Figure 5. IPU-derived degradation products in (A) shoots and (B) roots and conjugates of IPU in (C) shoots and (D) roots extracted from samples from wheat treated with IPU for 10 days under MeJA treatment or no treatment. Seedlings grew in soils with IPU (4 mg kg−1) and without IPU (CK, control) for 4 days. After that, the leaves were sprayed with 0.1 μmol L−1 MeJA once a day for 6 days. Values are means ± standard deviations (n = 3). Asterisks indicate the significant difference between the treatments with IPU and MeJA and treatments with IPU (p < 0.05).

Figure 6. Proposed metabolic pathway of IPU in wheat exposed to IPU and/or MeJA. The contents in the green box are metabolized from IPU. The substances in the red box are conjugates conjugated with an amino acid of IPU. The contents in the blue box are conjugates conjugated via glycosylation of IPU.

[REFERENCES]
(1) Sørensen, S. R., Bending, G. D., Jacobsen, C. S., Walker, A., and Aamand, J. (2003) Microbial degradation of isoproturon and related phenylurea herbicides in and below agricultural fields. FEMS Microbiol. Ecol. 45, 1−11.
(2) Song, N. H., Yin, X. L., Chen, G. F., and Yang, H. (2007) Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils. Chemosphere 68, 1779−1787.
(3) Seiber, J. N., and Kleinschmidt, L. A. (2011) Contributions of pesticide residue chemistry to improving food and environmental safety: past and present accomplishments and future challenges. J. Agric. Food Chem. 59, 7536−7543.
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