Article pubs.acs.org/JAFC
Variable Levels of Glutathione S‑Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots Dongzhi Li,† Li Xu,† Sen Pang,† Zhiqian Liu,§ Kai Wang,† and Chengju Wang*,† †
College of Science, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Haidian District, Beijing 100193, People’s Republic of China § Department of Economic Development, Jobs, Transport and Resources, AgriBio, 5 Ring Road, Bundoora, Victoria 3083, Australia S Supporting Information *
ABSTRACT: Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor−glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots. KEYWORDS: maize, shoots and roots, herbicide tolerance, glutathione S-transferases, metolachlor
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INTRODUCTION
crop tolerance was also related to selective promotion of GST activity in crops.9−11 Although the relationship between GST activity and the tolerance of plant to herbicide has been extensively investigated, most studies characterized the expression of a single GST gene. Systematic analyses of the entire GST family are rare. McGonigle et al. reported that the substrate specificity of GSTs is quite broad and suggested that GST-mediated herbicide detoxification may not be the function of a single GST gene. Instead, it was suggested that this detoxification may be reflective of the expression characteristics and the functionality of all of the GSTs present in a given species.12 In the present study, the expression profiles of the entire GST gene family in roots and shoots of maize seedling were compared. The effects of a GST inhibitor on maize growth and GST activity, as well as the in vitro herbicide−glutathione conjugation were assessed. These experiments collectively revealed the direct role of GSTs in the differential tolerance to herbicide. The results of this study will lead to a better understanding of the molecular mechanisms of differential tolerance to metolachlor between maize shoots and roots.
Maize (Zea mays L.) is a widely planted food crop around the world. Chloroacetamides are commonly applied to maize as pre-emergence herbicides for the control of annual grasses and some broad-leaved weeds.1 Metolachlor, one of the most extensively used chloroacetamide herbicides in maize, can cause crop damage if applied improperly, and differential tolerance to this herbicide was also observed across different maize cultivars.2 Therefore, it is important to understand the mechanism of crop tolerance to this herbicide. This will permit more rational use and enable the design of a breeding program to obtain high-tolerance cultivars. Plants have versatile defense systems to mitigate the toxic effects of a wide range of natural and synthetic compounds present in the environment. Glutathione S-transferases (GSTs, EC 2.5.1.18) are among such defense systems. Their role in herbicide detoxification has been widely investigated during the previous decades.3,4 GSTs catalyze the nucleophilic addition of reduced glutathione (GSH) to the electrophilic groups of hydrophobic toxic molecules, forming water-soluble and inactive conjugates.5,6 Multiple studies have revealed the involvement of GSTs in herbicide selectivity and differential tolerance between different biotypes of the same crop or weed species. For example, Hatton et al. reported that the expression levels of four GSTs in Setaria faberi were 20-fold lower than those in maize.7 Reade and Cobb found that the resistant blackgrass biotype had approximately doubled GST activity compared with the susceptible biotype.8 Safeners are agrochemicals that increase crop tolerance to herbicides. Several studies have revealed that, when using safeners, the enhanced © XXXX American Chemical Society
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MATERIALS AND METHODS
Chemicals. The herbicide metolachlor (97% purity) was supplied by the Key Laboratory of Pesticide Chemistry and Application Technology, China Agricultural University. The GST model substrate, Received: Revised: Accepted: Published: A
September 16, 2016 December 14, 2016 December 19, 2016 December 19, 2016 DOI: 10.1021/acs.jafc.6b04129 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Metolachlor Conjugation Assay. The conjugation rates of metolachlor with GSH were measured in vitro by high-performance liquid chromatography (HPLC) using crude enzyme as previously described by Edwards et al.14 The enzyme preparation (120 μL, adjusted to 1 mg mL−1) was added to 50 μL (0.1 mol L−1) of potassium phosphate buffer (pH 6.8). The mixture was then transferred to a water bath at 37 °C, and 10 μL of metolachlor solution (10 mmol L−1 dissolved in acetone) was added, immediately followed by the addition of 20 μL of freshly prepared reduced glutathione (100 mmol L−1) adjusted to pH 7.0 with 0.1 mol L−1 NaOH. After incubation for 60 min, the reaction was stopped by the addition of 10 μL of HCl (3 mol L−1) and was allowed to stand on ice for 30 min before removal of precipitated protein by centrifugation (12000g, 5 min). Boiled enzyme was used as a control to correct for the nonenzymatic reactions. The residual metolachlor in the supernatant was analyzed by using an Agilent 1260 HPLC system equipped with a reversed-phase C18 analytical column (4.6 mm × 150 mm, 3.5 μm) kept at 25 °C. The mobile phase was acetonitrile/water (60:40, v/v) containing 0.1% acetic acid at a flow rate of 1 mL min−1. The injection volume was 20 μL. Metolachlor was quantified by using a UV detector at 230 nm. The relative detoxification rate was calculated by measuring the reduction of metolachlor. GST Gene Expression Analysis. The harvested shoots and roots from maize seedlings treated with 30 μmol L−1 metolachlor for 96 h were ground into fine powder in liquid nitrogen. Total RNA was isolated using RNAprep pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocols. First-strand cDNA was synthesized from 1 μg total RNA using the Fast Quant RT Kit (Tiangen Biotech) in accordance with the manufacturer’s recommendations. Quantitative real-time PCR (qRT-PCR) was carried out using SYBR Green PCR Master Mix reagent kits (Tiangen Biotech) by an ABI Prism7500 Real-Time PCR System (Applied Biosystems by Life Technologies, Foster City, CA, USA). Reactions were run in a 20 μL mixture containing 10 μL of 2× SuperReal PreMix solution, 0.4 μL of 50× ROX Reference Dye, 0.6 μL of each of the forward and reverse primers (10 μmol L−1), 1 μL of cDNA template, and 7.4 μL of ddH2O. The amplification procedure was as follows: 95 °C for 15 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. Primers for the GSTs were designed on the basis of the mRNA sequences of maize using Primer Premier 5.0 software and are summarized in Table S1 (Supporting Information). Reference genes 18S rRNA (F, 5′GCTCTTTCTTGATTCTATGGGTGG-3′; R, 5′-GTTAGCAGGCTGAGGTCTCGTTC-3′) and GAPDH (F, 5′-CCATCACTGCCACACAGAAAAC-3′; R, 5′-AGGAACACGGAAGGACATACCAG-3′) were used as internal controls to normalize the amount of transcript among different samples. The primer specificity was checked by a melt curve analysis, and the amplification efficiency was estimated using the equation E = 10−1/slope, where the slope was derived from the plot of the amplification cycle time (Ct value) versus serially diluted template cDNA. The relative expression levels of genes were calculated by using the 2−ΔΔCT method.18 Three biological replicates and three technical replicates were performed for each sample. Statistical Analysis. All statistical analyses were performed by using SPSS 16.0 (SPSS, Chicago, IL, USA). Differences in GST gene expression levels between maize tissues were analyzed by Student’s t test. The results of GST activity and metolachlor−glutathione conjugation rates were subjected to one-way analysis of variance (ANOVA), combined with Duncan’s post hoc comparison test (p < 0.05 was considered significant).
1-chloro-2,4-dinitrobenzene (CDNB), was purchased from J&K (Beijing, China) and the GST inhibitor, ethacrynic acid (ETA), from Sigma-Aldrich (Beijing, China). Plant Materials and Treatment. Maize (Z. mays cv. Zhengdan958) seedlings were used to study the mechanism of differential tolerance to metolachlor between shoots and roots. Maize kernels were soaked in distilled water for 12 h at room temperature and then allowed to germinate on moist cheesecloth for 36−48 h in an intelligent artificial climate chamber RXZ-3808 (Jiangnan Instrument, Ningbo, China) at 28 °C, 75% relative humidity, and 16/8 h day/night cycle. Plastic pots filled with sterile sand (160 °C/3 h) were used as a culture medium. Sixty milliliters of metolachlor at variable concentrations was applied to each pot before sowing of pregerminated kernels. An equal volume of water was applied for the control treatment. Each treatment contained three pots (six plants per pot). The plants were maintained in the same growth chamber with the same growing conditions for the duration of the experiment. For the metolachlor dose−response test, the herbicide was applied at 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 μmol L−1. After 96 h (seedlings at the second-leaf stage), the lengths of the shoots and roots of each plant were measured and the toxicity of metolachlor was assessed using IC50. For the GST enzymatic assay and GST gene expression analysis, metolachlor was applied at a fixed concentration of 30 μmol L−1. Shoots and roots of maize seedlings were harvested separately 96 h after treatment and frozen in liquid nitrogen. The plant samples were either assayed immediately or stored at −80 °C until use. GST Inhibition in Relation to Maize Growth. We tested the effect of a GST enzyme inhibitor, ETA, on the toxicity of metolachlor to maize seedlings. The plant growing conditions were the same as previously described. Seven concentrations of ETA (0, 50, 100, 200, 400, 800, and 1600 μmol L−1) alone were applied in the first instance to determine the maximal tolerated dosage. Metolachlor at 30 μmol L−1 was then applied in combination with ETA (800 μmol L−1). The effect of metolachlor on growth inhibition in the presence of ETA was measured 96 h after herbicide application. GST Activity Assay and Effect of ETA. The extraction of GST enzymes was performed according to the protocols described by Fuerst et al.13 and Edwards and Dixon14 with some modifications. The harvested shoots and roots were ground into fine powder in liquid nitrogen and homogenized in the extraction buffer (1:5, w/v) containing 0.1 mol L−1 Tris-HCl (pH 7.5), 2 mmol L−1 EDTA, 1 mmol L−1 DTT, and 50 g L−1 polyvinyl polypyrrolidone (PVPP). The homogenate was filtered through eight layers of cheesecloth and then centrifuged at 12000g for 10 min. The supernatant was used to assay the activity of GST enzymes. Unless otherwise specified, all steps were carried out at 4 °C. GST activity toward the substrate CDNB was assayed spectrophotometrically by measuring the change of A340 according to protocols described by Irzyk and Fuerst.15 The assay mixture consisted of 2 mL of 0.1 mol L−1 potassium phosphate (pH 7.0), 50 μL of glutathione (7.5 μmol L−1), 100 μL of CDNB (2.5 μmol L−1), and 20 μL of enzyme extract, which had been adjusted to identical concentrations (1 mg mL−1 of protein). Reactions were initiated by the addition of CDNB, and A340 was monitored for 90 s in a time-driver model. The activity of GSTs was calculated using the equation GST activity (nmol min−1) = (ΔOD340 × V)/(ε × L), where ΔOD340 represents the absolute value of A340 per minute, V is the volume of the reaction system in milliliters, ε (9.6 mM−1 cm−1) is the extinction coefficient of the CDNB−GSH conjugate, and L is the cuvette path in centimeters. The GST activity values were corrected for nonenzymatic conjugation. Inhibition of enzymatic activity by ETA was measured by the same reaction system with 10 μL of ETA added before CDNB. The concentration of ETA required for 50% inhibition (IC50) was determined from a plot of residual activity against inhibitor concentration.16 Protein was quantified as previously described by Bradford using bovine serum albumin as the reference protein.17 All of the experiments were conducted in triplicate, and representative results are shown.
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RESULTS Differential Tolerance to Metolachlor between Shoots and Roots. On the basis of our bioassay results, a clear difference in tolerance to metolachlor was observed between shoots and roots of maize cultivar Zhengdan958. The growth of the shoots was inhibited in a concentrationdependent manner. However, the roots did not show any appreciable change regardless of herbicide concentrations (Figure 1). The IC50 values for shoots and roots were 42.3
B
DOI: 10.1021/acs.jafc.6b04129 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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of tissue (Table 1). ETA suppressed the activity of GSTs extracted from both roots and shoots in a concentrationTable 1. GST Activity in Maize Shoots and Roots GST activitya (nmol/min/μg) concentration of metolachlor (μmol L−1)
shoots
roots
0 30
0.22 ± 0.02a 0.21 ± 0.03a
1.61 ± 0.10b 1.67 ± 0.13b
a Mean values of three replicates (±standard deviations); values followed by a different letter are significantly different (p < 0.05).
Figure 1. Differential tolerance between shoots and roots of maize seedlings to metolachlor. Numbers 0, 25, 50, 100, and 200 represent metolachlor concentrations (in μmol L−1).
dependent manner (Figure 3); the IC50 values of ETA for GST activity were 66.2 and 281.0 μmol L−1 for shoots and roots, respectively.
and >200 μmol L−1, respectively, indicating an at least 5-fold difference in metolachlor tolerance between the shoots and roots of maize. Effect of a GST Inhibitor on the Toxicity of Metolachlor. To ascertain that GST enzymes are implicated in the contrasting response of shoots and roots to herbicide treatment, we tested the inhibitory effect of metolachlor in the presence of a GST inhibitor, ETA. Figure 2A shows that the
Figure 3. Inhibition of GST activity by ETA.
Metolachlor Conjugation Assay Results. We sought to reveal whether the higher activity of GSTs in roots compared to shoots could result in an enhanced herbicide detoxification. To do so, we measured the in vitro conjugation rate of metolachlor with GSH using crude enzyme extracts from shoots and roots. Figure 4 shows that GST-mediated conjugation of metolachlor with GSH was 2-fold faster in root extract compared to shoot
Figure 2. Effect of a GST inhibitor, ETA, on the growth of maize shoots and roots: (A) ETA applied alone at 0, 50, 100, 200, 400, 800, and 1600 μmol L−1; (B) metolachlor applied alone at 30 μmol L−1 (M) and in combination with ETA at 800 μmol L−1 (M+ETA).
maximal tolerated dose of ETA applied alone is 800 μmol L−1. When ETA (800 μmol L−1) was applied together with metolachlor at 30 μmol L−1, a synergistic effect on growth inhibition was observed for both shoots and roots (Figure 2B). In the absence of ETA, metolachlor at 30 μmol L−1 was well tolerated by maize seedlings. However, when metolachlor was combined with ETA, a severe toxicity was observed for both roots and shoots, implying that GST activity plays a role in mitigating the toxic effect of the herbicide, especially on roots. GST Activity Assay and Effect of an Inhibitor. The intrinsic activity of GSTs in roots was approximately 8-fold that in shoots in nontreated plants, and the herbicide treatment did not cause any significant change in GST activity in either type
Figure 4. In vitro conjugation rate of metolachlor with glutathione catalyzed by crude enzyme extract from maize shoots and roots. CK and 30 denote control and 30 μmol L−1 metolachlor-treated samples, respectively. Error bars indicate standard deviation (n = 3). Columns with different letters are significantly different (using one-way analysis of variance, combined with Duncan’s post hoc comparison, p < 0.05). C
DOI: 10.1021/acs.jafc.6b04129 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Expression profiles of GST genes in shoots and roots: (A) comparison of GST gene expression in nontreated shoots and roots (shoots data are normalized to 1); (B) effect of metolachlor on the expression of GST genes in shoots (nontreated shoots data are normalized to 1); (C) effect of metolachlor on the expression of GST genes in roots (nontreated roots data are normalized to 1); (D) comparison of GST gene expression in shoots and roots after metolachlor treatment (shoots data are normalized to 1). RQ represents the relative quantity of GST gene transcript. Error bars are standard deviation (n = 3). Stars indicate significant difference (analyzed by Student’s t test, p < 0.05).
extract. Again, no significant difference was found between control and herbicide-treated samples for both roots and shoots. Expression Profiles of GST Genes in Shoots and Roots. The expression profiles of all members of the maize GST gene family in control and herbicide-treated roots and shoots were compared. Among the 42 members of the maize GST gene family, the expression levels of 7 genes (GST19, GST21, GST27, GST29, GST34, GST38, and GST40) were too low to be accurately determined (Ct > 35). Consequently, a total of 35 GST genes were analyzed in this study. The expression profiles of the 35 GST genes in shoots and roots of Zhengdan958 are summarized in Figure 5. In nontreated samples, the expression level of five GST genes (GST11, GST12, GST13, GST14 and GST6) was approximately 100-fold higher in roots than in shoots. Many of the remaining genes were 5−10-fold higher in roots compared to shoots (Figure 5A). Only three genes (GST10, GST24, and GST33) showed higher expressions in shoots. The effect of metolachlor on the expression of GST genes was tissue specific. The
expression of 18 GST genes in shoots was significantly downregulated, whereas that of 9 GST genes was up-regulated after a metolachlor treatment for 96 h (Figure 5B). In the case of roots, the metolachlor treatment suppressed the expression of 11 GST genes, but induced that of 10 GST genes (Figure 5C). The relative expression levels of most GST genes in roots versus shoots were not changed after the herbicide treatment, and higher expression of all but two genes in roots was still observed (Figure 5D).
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DISCUSSION
The differential tolerance to metolachlor between roots and shoots of maize seedlings is an interesting finding, given that the root system is directly exposed to soil-applied herbicide. Understanding the mechanisms underlying this observation is important for a safe application of this widely used herbicide. As GSTs are known to be involved in herbicide detoxification, our first step focused on the comparative study of GST activity and GST gene expression in shoots and roots. D
DOI: 10.1021/acs.jafc.6b04129 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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We have found that GST activity in roots is approximately 8fold higher than that in shoots, which coincides with the higher tolerance of roots to metolachlor. However, more evidence was needed to confirm the involvement of GSTs in the contrasting herbicide tolerance between shoots and roots. ETA is a known GST inhibitor and has been used in several studies aiming to assess the effect of it on GST activity.16,19 In this study we have shown that by suppressing GST activity with ETA, maize roots become more sensitive to the herbicide. This is direct proof that GSTs are associated with the elevated herbicide tolerance in roots. An in vitro enzymatic assay on herbicide−glutathione conjugation further indicated that GST-mediated herbicide transformation is correlated with metolachlor tolerance in different parts of maize seedlings. On the basis of the nomenclature of plant GSTs, maize GSTs can be classified into three classes, namely, phi, zeta, and tau.5 Phi and tau GSTs are dominant, plant-specific classes, which are capable of catalyzing the conjugation of chloroacetanilide herbicides.20,21 GSTI, GSTIII, and GSTIV belong to the phi class, which can enhance the tolerance to chloroacetanilide herbicides when overexpressed in transgenic crops.13,22,23 A clear function of GSTIV in protecting tobacco plants from metolachlor injury has also been observed.24 GST5, GST6, and GST7, which are from the tau class, were also reported to be involved in herbicide detoxification, especially GST5 and GST7, which exhibited activity toward metolachlor in vitro.21,25 As reported by McGonigle et al., 80% of maize phi-class GSTs are made up of a combination of GSTI (42%), GSTIII (13%), and GSTIV (24%), whereas 26% of maize tau-class GSTs are GST5.12 In our study, the expression of all of these genes was 5−10-fold higher in roots compared to shoots with or without metolachlor treatment. Therefore, the function of GSTI, GSTIII, GSTIV, and GST5 may be the driving force for the difference in metolachlor tolerance between shoots and roots. After metolachlor treatment, the expressions of GSTI, GSTIII, GSTIV, and most of tau-class members were decreased in shoots, whereas no significant change in the expressions of GSTI, GSTIII, and GSTIV and an increased expression of some tau-class members were observed in roots. This is in agreement with the modest change in overall GST activity after herbicide treatment. Indeed, similar results were reported by Sari-Gorla et al., who found that GST activity was increased by alachlor only in tolerant, but not in susceptible, maize lines.26 A correlation of herbicide tolerance with GST activity, rather than glutathione content, was found in a range of crops and weeds.27 Rossini et al. reported that the GST activity had a strong correlation with alachlor tolerance in three maize inbred lines.28 Reade et al. also found a strong correlation between the resistance ratio of black-grass and GST activity.8 In the present study, we have found that the total activity of GSTs is much higher in roots compared to shoots. This result is consistent with the findings of Sari-Gorla et al., who compared GST activity across different tissues, such as roots, leaves, and pollen, in 15 maize inbred lines.26 Therefore, the total GST activity appears to be a reliable indicator for metolachlor tolerance in maize tissues. In conclusion, our study demonstrated that the difference in tolerance to metolachlor between maize shoots and roots is correlated with the variable levels of GST activity. This enhanced GST activity in roots appears to result from a higher level of expression of genes in the GST family.
Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04129. Primers of GST genes (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(C.W.) E-mail:
[email protected]. Phone: +86 (0)10 62733924. Fax: +86 (0)10 62734294. ORCID
Dongzhi Li: 0000-0002-1245-9244 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Weisong Zhao and Yu Mei for their kind help in conducting the experiment and Mingqi Zheng and Jiazhen Jiang for their technical support during this study.
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REFERENCES
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