Involvement of antioxidant capacity in quinclorac tolerance in Eleusine indica

Environmental and Experimental Botany 74 (2011) 74–81

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Involvement of antioxidant capacity in quinclorac tolerance in Eleusine indica Yukari Sunohara ∗ , Shinjiro Shirai, Hiroki Yamazaki, Hiroshi Matsumoto Doctoral Program in Life Sciences and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

a r t i c l e

i n f o

Article history: Received 29 September 2010 Received in revised form 2 May 2011 Accepted 3 May 2011 Keywords: Antioxidant capacity Auxinic herbicide Digitaria adscendens Eleusine indica Reactive oxygen species Superoxide anion Quinclorac Quinclorac-tolerance

a b s t r a c t The relationship between quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) tolerance and scavenging capacity of reactive oxygen species (ROS) in Eleusine indica was investigated to understand the tolerance mechanisms of E. indica to the herbicide. E. indica was approximately 104-fold more tolerant to quinclorac than Digitaria adscendens based on GR40 (herbicide dose required to cause a 40% reduction in plant growth) values determined 6 days after treatment. Quinclorac (10 ␮M) induced the overproduction of ROS (presumably superoxide anion (O2 − )) in the root tips of D. adscendens 24 h after treatment. On the other hand, 10 ␮M quinclorac did not induce the ROS production in the roots of E. indica. The inherent superoxide anion scavenging activity (SOSA) was 4.2-fold higher in E. indica than that in D. adscendens. The constitutive activities of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) were 1.9, 4.7, and 3.0 times higher, respectively in E. indica than those in D. adscendens. Exogenously applied H2 O2 , one of the main ROS, decreased the chlorophyll content in leaf discs of E. indica and D. adscendens. However, the chlorophyll content in E. indica was much higher than that in D. adscendens, indicating that E. indica with its high antioxidant capacity can alleviate H2 O2 -induced phytotoxicity and is more tolerant to H2 O2 than D. adscendens. These results suggest that the high scavenging capacity of ROS in E. indica could be one factor in its tolerance to quinclorac. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) is a highly selective auxinic herbicide and is mainly used in rice paddies and turf fields to control some certain broad-leaved weeds and important grass weeds, such as Echinochloa, Digitaria, and Setaria species (Menck et al., 1985; Wuerzer and Berghaus, 1985; Chism et al., 1991; Grossmann and Kwiatkowski, 2000). There have been many reports concerning the action of quinclorac in grass species (Berghaus and Wuerzer, 1987; Koo et al., 1991, 1996; Grossmann and Kwiatkowski, 1993, 1995, 2000; Grossmann and Scheltrup, 1997; Grossmann, 1998; Sunohara and Matsumoto, 1997, 2004, 2008; Sunohara et al., 2003a,b; Sunohara et al., 2010; Tresch and Grossmann, 2003a,b; Abdallah et al., 2006). Quinclorac acts by stimulating the induction of 1aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14), a key enzyme of ethylene biosynthesis (Grossmann and Scheltrup, 1997; Grossmann, 1998). As a result, ethylene biosynthesis is increased and the tissues accumulate cyanide, an ethylene co-product formed during the oxidation of ACC. This cyanide accumulation occurs in sensitive species but not in tolerant species (Grossmann, 1998; Grossmann and Kwiatkowski, 2000).

∗ Corresponding author. Tel.: +81 29 853 4902; fax: +81 29 853 4605. E-mail address: [email protected] (Y. Sunohara). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.001

The accumulation of cyanide resulting from quinclorac-induced ethylene production has been proposed as the main mechanism of action of this herbicide in susceptible grasses (Grossmann and Kwiatkowski, 1995, 2000; Abdallah et al., 2006). We reported previously that quinclorac also induced oxidative injury in Echinochloa oryzicola Vasing. (Sunohara and Matsumoto, 2004), Digitaria adscendens (H.B.K.) Henr. (Sunohara et al., 2010), and maize (Sunohara and Matsumoto, 2008), a susceptible grass species. In addition, quinclorac induced overproduction of reactive oxygen species (ROS) (presumably superoxide anions (O2 − )) in maize roots (Sunohara and Matsumoto, 2008). These results suggest that ROS are additionally involved in quinclorac-induced phytotoxicity in grass species. Eleusine indica (L.) Gaerth. and D. adscendens (H.B.K.) Henr. are summer annual C4 grasses with tussocky, prostrate growth forms. They are distributed throughout the world and have sympatric distributions in the North Kanto district of eastern Japan (Takematsu and Ichizen, 1997), and their morphological characteristics, life forms, and phenological schedules are similar. They can be controlled by some turf herbicides, but it has been observed that the turf herbicide quinclorac cannot control E. indica in some field experiments, while it can efficiently suppress D. adscendens, indicating that their sensitivities to quinclorac are totally different. Zawierucha and Penner (2000) reported that quinclorac selectivity between quinclorac-tolerant E. indica and quinclorac-sensitive D. sanguinalis L. was not adequately explained by differential

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absorption, translocation, metabolism, or foliar spray retention. Therefore, the large difference in tolerance of E. indica and D. adscendens to quinclorac may also be related to the target site. In our previous study (Sunohara et al., 2010), quinclorac (≤10 ␮M) had no effects on the seedling growth and chlorophyll content in E. indica, but 100 ␮M 2,4-dichlorophenoxyacetic acid (2,4-D), another auxinic herbicide, inhibited seedling growth and slightly reduced chlorophyll content in the plants. In D. adscendens, seedling growth was inhibited by both quinclorac (1–100 ␮M) and 2,4-D (100 ␮M), and 10 ␮M quinclorac greatly reduced the chlorophyll content and markedly induced lipid peroxidation; whereas 100 ␮M 2,4-D did not affect the chlorophyll content and lipid peroxidation. In ethylene production, quinclorac (10 ␮M) did not induce production in E. indica but did so in D. adscendens seedlings. Interestingly, 2,4-D (100 ␮M) rapidly and strongly induced ethylene production in both E. indica and D. adscendens seedlings, and the level of 2,4-D-induced ethylene in E. indica was much higher than that in D. adscendens. That is, ethylene production was unlikely to be enhanced specially by quinclorac in E. indica. Application of 1aminocyclopropane-1-carboxylic acid, an intermediate of ethylene biosynthesis, reduced the growth of E. indica and D. adscendens. These results suggest that the failure of activation of ethylene biosynthesis by quinclorac may be an important factor for quinclorac tolerance in E. indica. Moreover, these results also suggest that quinclorac has additional oxidative actions related to chlorosis and lipid peroxidation, and quinclorac tolerance in E. indica may be a result of multiple factors, such as the non-activation of ethylene biosynthesis by the herbicide and anti-oxidation-related factors. However, the involvement of anti-oxidation-related factors in quinclorac tolerance in E. indica has remained unclear. Therefore, the present study aims to investigate whether the scavenging capacity for ROS is one factor in quinclorac tolerance in E. indica, by testing the hypothesis that the differential scavenging capacity for ROS may be related to the large difference in sensitivity of E. indica and D. adscendens to quinclorac. Moreover, this study may provide basic information for the importance of scavenging capacity for ROS in the tolerance to oxidative stress caused by various environmental stresses. 2. Methods 2.1. Plant materials Germinated seeds of E. indica and D. adscendens were grown hydroponically in Kasugai nutrient solution (Ohta et al., 1970) in a growth chamber at 25/20 ◦ C with 14 h of light (250–300 ␮mol m−2 s−1 ) per day. Seedlings of E. indica and D. adscendens at the 3-leaf stage were used for all experiments. 2.2. Herbicide treatment and growth measurement Quinclorac (purity > 99%) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The roots of intact seedlings of each species were immersed in 0.01, 0.1, 1, 10, or 100 ␮M quinclorac solution containing 0.8% acetone and 0.2% dimethyl sulfoxide (DMSO) at 25 ◦ C for 12 h in the dark. The roots of untreated control seedlings were immersed in distilled water containing 0.8% acetone and 0.2% DMSO. After herbicide treatment, the roots were washed with distilled water and transplanted to herbicide-free nutrient solution in the growth chamber at 25/20 ◦ C with 14 h of light (250–300 ␮mol m−2 s−1 ) per day. The seedlings were washed with distilled water and then fresh weights (FW) of whole seedlings were measured at 6 days after treatment. A log–logistic analysis was used to describe the herbicide doseresponse relationships (Seefeldt et al., 1995; Schabenberger et al.,

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1999). The mathematical expression relating the response y to dose x is:



y = f(x) = ı +

˛−ı 1 + exp[ˇ (ln(x/GR50 )]



,

where ı = the lower limit, ˛ = the upper limit, ˇ = the slope, and GR50 = the dose giving a 50% reduction in plant growth. 2.3. Detection of ROS ROS production was estimated using a superoxide anion (O2 − )specific indicator, DHE, in accordance with the procedures of Yamamoto et al. (2002) with a minor modification. Root segments (1.0 cm length from the tip) of each species were excised from intact seedlings treated with 0 or 10 ␮M quinclorac or 100 ␮M 2,4-D 1 and 2 days after treatment, and then stained with 1 ␮M DHE in 100 ␮M CaCl2 , pH 4.75, by shaking gently for 2 h at room temperature in the dark. After washing the root segments with distilled water to remove residual dye, the roots were observed using a fluorescence microscope (Nikon E600 with a B-2A filter [excitation 450–490 nm, emission ≥520 nm]; Nikon, Tokyo, Japan). Fluorescence of ethidium derived from DHE oxidation by ROS (presumably O2 − ) was observed using the microscope. 2.4. Determination of SOSA ESR analysis was performed using a Model JES-FR30EX free radical monitor (JEOL Ltd., Tokyo, Japan). Manganese oxide provided a constant signal intensity, to which all peak heights were compared: the sample peak height divided by the peak height of MnO to give the relative peak height. ESR measurements were conducted under the following conditions: microwave power, 4.0 mW; amplitude, 2.5 × 100; time constant, 0.1 s; magnetic field, 336.1 mT; modulation width, 0.63 × 0.1 mT; sweep time, 2 min. Two hundred milligrams of excised shoots of E. indica or D. adscendens was crushed in liquid nitrogen and homogenized with 5 ml of 0.1 M potassium phosphate buffer (pH 7.4). After centrifugation at 6000 × g for 10 min at 4 ◦ C, the supernatant was filtered through Miracloth® (Calbiochem, San Diego, US). The filtrate was then diluted four times with 0.1 M potassium phosphate buffer (pH 7.4), and it was used as a sample solution for the determination of SOSA by an ESR-spin trapping method. Spin-trapping methods for superoxide radicals were based on the methods of Rosen and Freeman (1984). Superoxide anions were generated by a An hypoxanthine (HPX)–xanthine oxidase (XOD) reaction system. A solution of 15 ␮L of 8.97 M 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Labotec, Tokyo, Japan) as a spin-trapping agent, 50 ␮L of 2 mM HPX, 35 ␮L of 5.5 mM diethylenetriamine-N,N,N ,N ,N -pentaacetic acid (DTPA), 50 ␮L of sample solution, and 50 ␮L of XOD (0.4 units ml−1 ) was prepared in a 0.1 M potassium phosphate buffer (pH 7.4). This was then transferred to an ESR spectrometry flat cell, and the DMPO–OO− spin adduct was quantified for 1 min after the addition of XOD. SOSA was expressed as superoxide dismutase (SOD) equivalent units per gram fresh weight. 2.5. Extraction and activity assays of antioxidative enzymes 2.5.1. Enzyme extraction Five hundred milligrams of excised shoots of E. indica and D. adscendens was crushed in liquid nitrogen and homogenized with 5 ml of 25 mM potassium phosphate buffer (pH 7.8) containing 0.4 mM EDTA–4H, 1 mM ascorbic acid (AsA), and 2% polyvinyl polypyrrolidone (PVPP). After centrifugation at 15000 × g for 20 min at 4 ◦ C, the supernatant was filtered through Miracloth®

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(a) Eleusine indica

120

120 100 FW (% of control)

FW (% of control)

100

(b) Digitaria adscendens

80 60 40

GR40 = 198.1 μM GR50 > 1000 μM

20

0.1

1

10

100

60 40 20

0 0.01

80

1000

Quinclorac concentration (μM)

0 0.01

GR40 = 1.9 μM GR50 = 3.2 μM 0.1

1

10

100

Quinclorac concentration (μM)

Fig. 1. Effects of quinclorac on seedling growth of (a) Eleusine indica and (b) Digitaria adscendens after 12 h root treatment. The fresh weights of whole plants were measured at 6 days after treatment. The mean fresh weight of non-treated-control E. indica and D. adscendens were 0.06 ± 0.01 g 3 seedlings−1 and 0.39 ± 0.03 g 3 seedlings−1 , respectively. GR40 : herbicide dose required to cause a 40% reduction in plant growth. GR50 : herbicide dose required to cause a 50% reduction in plant growth.

(Calbiochem). The filtrate was then used as an enzyme extract for catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2) assays. 2.5.2. Enzyme assays CAT activity was assayed in a 1 ml reaction mixture containing 0.95 ml of 50 mM potassium phosphate buffer (pH 7.0, containing 10 mM H2 O2 ) and 0.05 ml of enzyme extract. The subsequent decomposition of H2 O2 was observed at 240 nm (E = 0.0394 mM−1 cm−1 ) (Aebi, 1983). APX activity was determined by the method of Nakano and Asada (1987). The activity was assayed in a 1 ml reaction mixture containing 0.25 ml of 100 mM potassium phosphate buffer (pH 7.0), 0.25 ml of 1 mM AsA, 0.25 ml of 0.4 mM EDTA–4H, 0.01 ml of 10 mM H2 O2 , 0.19 ml of distilled water, and 0.05 ml of enzyme extract. The subsequent decrease in AsA was observed at 290 nm (E = 2.8 mM−1 cm−1 ). GR activity was determined by the method of Halliwell and Foyer (1978). Its activity was assayed in a 1 ml reaction mixture containing 0.25 ml of 100 mM potassium phosphate buffer (pH 7.8), 0.05 ml of 10 mM oxidized glutathione (GSSG), 0.12 ml of 1 mM NADPH, 0.48 ml of distilled water, and 0.1 ml of enzyme extract. The resultant decrease in NADPH was observed at 340 nm (E = 6.1 mM−1 cm−1 ). 2.6. Effect of H2 O2 on chlorophyll content in leaves of E. indica and D. adscendens Leaf discs (5 mm in diameter) were taken from E. indica and D. adscendens seedlings at the 3.0-leaf stage. Three discs were placed in a 2-cm-diameter plastic petri dishes with 1.5 ml of 0, 100, 200, 500, or 1000 mM H2 O2 solution and soaked for 1 h in the dark. Then the petri dishes were exposed to light (4–7 ␮mol m−2 s−1 ) for 27 h at 25 ◦ C. After exposure to the light, the leaf discs were soaked in absolute DMSO (1 ml leaf disc−1 ) in a glass vial. The vial was capped tightly and incubated at 30 ◦ C for 48 h in the dark. The concentration of the extracted pigments (chlorophyll a and chlorophyll b) was calculated based on the absorbance values at 664 and 648 nm, in accordance with the method of Chappelle et al. (1992). 2.7. Statistical analyses All results were represented as the mean ± S.E. of at least three replicates, and all experiments were repeated at least once. For all statistical analyses, relationships were considered to be significant when P < 0.05. To estimate GR40 and GR50 values, the SPSS procedure was performed using a log–logistic model. The

effect of quinclorac on SOSA in each species was analyzed using the Tukey–Kramer test. The effects of quinclorac on the activities of CAT, APX, and GR in each species were analyzed using Student’s t-test. Differences in chlorophyll content between nontreated (control) and H2 O2 -treated leaf discs were also analyzed using Student’s t-test. 3. Results and discussion 3.1. Effects of quinclorac on the seedling growth of E. indica and D. adscendens The phytotoxic activities of quinclorac on the seedlings of E. indica and D. adscendens were estimated by the log–logistic method. GR40 (herbicide dose required to cause a 40% reduction in plant growth) values determined 6 days after treatment for E. indica and D. adscendens were 198.1 ␮M and 1.9 ␮M, respectively; a difference of 104-fold between E. indica and D. adscendens (Fig. 1). GR50 values for E. indica and D. adscendens were >1000 ␮M and 3.2 ␮M, respectively, indicating that E. indica was >313 times more tolerant than D. adscendens based on the GR50 values determined 6 days after treatment (Fig. 1). As we reported previously (Sunohara et al., 2010), quinclorac-treated D. adscendens seedlings showed chlorotic discoloration and necrosis that progressed from the growing areas of newly emerged leaves followed by reddish discoloration, wilting, and desiccation of the entire shoot. 3.2. Effects of quinclorac on ROS production in E. indica and D. adscendens ROS production was investigated by staining roots of E. indica and D. adscendens with dihydroethidium (DHE). DHE enters the cell and is oxidized by ROS to yield fluorescent ethidium. Ethidium binds to DNA (Eth-DNA), further amplifying its fluorescence (Carter et al., 1994). Fig. 2 shows the effects of 10 ␮M quinclorac on ROS production in roots of E. indica and D. adscendens 24 h after 12 h root treatment. In E. indica, fluorescence images did not substantially differ between quinclorac-treated and non-treated (control) roots (Fig. 2a), suggesting that ROS production was not enhanced by 10 ␮M quinclorac in E. indica roots. On the other hand, the fluorescence in 10 ␮M quinclorac-treated root tips of D. adscendens was obviously greater than that in control root tips (Fig. 2b). These results indicated that 10 ␮M quinclorac induced ROS production in the root tips of D. adscendens. Quinclorac-enhanced ROS production was also observed in the roots of maize, another susceptible grass species (Sunohara and Matsumoto, 2008). DHE is known to be relatively specific for O2 − (Becker et al., 1999). Therefore, the

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Fig. 2. Effects of 10 ␮M quinclorac on ROS production in root tips of (a) Eleusine indica and (b) Digitaria adscendens 24 h after 12 h root treatment. Bright fluorescence shows ROS production (presumably O2 − ). Scale bars indicate 500 ␮m. Fluorescence images were obtained using a Nikon E600 fluorescence microscope with a B-2A filter (excitation 450–490 nm, emission ≥520 nm).

quinclorac-induced increase in the fluorescence may be due to O2 − production. In this study, roots were used for the fluorescence observations because other fluorescence not due to ROS interrupted ROS observation in the shoots. In our previous studies, quinclorac also enhanced the formation of thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxidation, in the shoots of D. adscendens (Sunohara et al., 2010) and E. oryzicola Vasing. (Sunohara and Matsumoto, 2004). Therefore, quinclorac may cause overproduction of ROS not only in roots but also in shoots. There are many potential sources of ROS in plants, including intracellular sources such as mitochondria, chloroplasts and peroxisomes, cell wall peroxidase, plasma membrane NADPH oxidase, and amine oxidases and apoplastic oxalate oxidase (Grant and Loake, 2000; Mittler, 2002; Neill et al., 2002; Vranová et al., 2002). Because quinclorac induced ROS production in the roots of several susceptible grasses (Fig. 2b; Sunohara and Matsumoto, 2008), another source other than chloroplasts seem to be related to the mechanism of quinclorac-induced ROS overproduction. Further studies are needed to clarify the source of ROS production. As described above, quinclorac induced ROS (probably O2 − ) production in D. adscendens (Fig. 2b). O2 − is one of the major ROS

among the free radicals that are generated first after oxygen is taken into living cells. O2 − changes to other harmful ROS and free radicals such as H2 O2 and hydroxyl radicals. Excessive ROS seriously disrupt normal metabolism through oxidative damage to lipids, proteins and nucleic acids (Fridovich, 1986; Davies, 1987; Imlay and Linn, 1988). Therefore, quinclorac-induced O2 − overproduction could be one factor in phytotoxicity by the herbicide. 3.3. SOSA in E. indica and D. adscendens seedlings SOSA were compared between E. indica and D. adscendens using ESR spectroscopy (Fig. 3a and b) because the involvement of O2 − overproduction in quinclorac-induced phytotoxicity was suggested. The mean values of SOSA were 1423.2 ± 158.8 and 336.0 ± 17.5 units g FW−1 in shoots of E. indica and D. adscendens 0 days after 12 h root treatment, respectively (Fig. 3a and b); the inherent scavenging activity was 4.2-fold higher in E. indica than that in D. adscendens. In plants, there are two kinds of antioxidative systems: (1) systems involving antioxidative enzymes such as SOD, glutathione peroxidase, CAT, and APX and (2) systems that produce antioxidants (AsA, ␣-tocopherol, ␤-carotene, cysteine, and ubiquinone, etc.) (Niki, 1991; Gupta et al., 1993; Casano et al., 1997;

Fig. 3. Effects of quinclorac on superoxide anion scavenging activity (SOSA) in shoots of (a) Eleusine indica and (b) Digitaria adscendens after 12 h root treatment. Bars with the same letter are not significantly different, P ≥ 0.05.

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Fig. 4. Effects of quinclorac on catalase (CAT) activity in shoots of (a) Eleusine indica and (b) Digitaria adscendens after 12 h root treatment. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.01 (control vs. quinclorac treatment).

Noctor and Foyer, 1998). In the ESR method used to quantify SOSA in the present study, AsA and anthocyanin mainly contribute to the value of SOSA, although the contribution of AsA and anthocyanin to SOSA differs among species and the amount of ROS (Nagata et al., 2003). Therefore, the inherent contents of AsA and anthocyanin may be high in E. indica, and the high contents may contribute to the high efficiency of eliminating O2 − in this species. Quinclorac (10 ␮M) did not affect SOSA in E. indica seedlings. However, the herbicide enhanced SOSA in D. adscendens seedlings 1 and 2 days after treatment, and it was 3.4 times higher in 10 ␮M quinclorac-treated seedlings than that in non-treated (control) seedlings 2 days after treatment. In the present study, quinclorac induced ROS (presumably O2 − ) generation in D. adscendens seedlings but not in E. indica seedlings 1 day after 12 h root treatment (Fig. 2). The synthesis of AsA and anthocyanin may be enhanced to remove quinclorac-induced excessive O2 − in D. adscendens. In fact, it was reported that the SOSA in ␥-rayirradiated-plants to produce ROS increased to ∼10 times the usual level with a concurrent increase in the storage of AsA and anthocyanin, indicating that ROS also activated the synthesis of AsA and anthocyanin (Nagata et al., 2003). However, the mean value (945.8 ± 30.0) of the induced activity to scavenge excessive O2 − 2 days after quinclorac treatment in D. adscendens was lower than the inherent scavenging activity (1423.2 ± 158.8) in E. indica. Under normal conditions, the degree of radical scavenging activity shown by AsA directly reflects the plant’s sensitivity and resistance to ROS stress induced by environmental conditions (Nagata et al., 2003). Therefore, the inherently high radical-scavenging ability in E. indica could contribute to tolerance to herbicides that induce ROS overproduction, such as quinclorac.

3.4. Antioxidative enzyme activities in E. indica and D. adscendens seedlings The activities of antioxidative enzymes, CAT, APX and GR, were compared between E. indica and D. adscendens (Figs. 4–6). The mean CAT activities were 296.9 ± 12.0 and 157.9 ± 26.5 ␮mol H2 O2 decomposed g FW−1 min−1 in shoots of E. indica and D. adscendens 0 days after 12 h root treatment, respectively (Fig. 4a and b); the constitutive level of CAT activity was 1.9-fold higher in E. indica than that in D. adscendens. Quinclorac (10 ␮M) induced CAT activity in E. indica seedlings 0 days after 12 h root treatment, but significant induction was not observed 2 days after treatment (Fig. 4a). In D. adscendens seedlings, the herbicide enhanced CAT activity from 2 days after treatment. The mean CAT activities were 108.1 ± 3.5 and 251.3 ± 24.8 ␮mol H2 O2 decomposed g FW−1 min−1 in control and 10 ␮M quinclorac-treated seedlings of D. adscendens, respectively; the CAT activity was 2.3-fold higher in quinclorac-treated D. adscendens seedlings than control seedlings. Quinclorac (10 ␮M) induced ROS (presumably O2 − ) production in the roots of D. adscendens 1 day after 12 h root treatment (Fig. 2b). O2 − is either spontaneously or enzymatically dismutated to H2 O2 via superoxide dismutase (SOD, EC 1.15.1.1) (Foyer et al., 1997). CAT catalyzes the disproportionation of H2 O2 to oxygen and water and is one of the principal H2 O2 -metabolizing enzymes in plant cells (Scandalios et al., 1997). Therefore, CAT activity may be induced to scavenge excessive H2 O2 in quinclorac-treated D. adscendens seedlings. The mean APX activities were 9.3 ± 0.5 and 2.0 ± 0.2 ␮mol AsA decomposed g FW−1 min−1 in shoots of E. indica and D. adscendens 0 days after 12 h root treatment, respectively (Fig. 5a and b); the inherent APX activity was 4.7 times higher in E. indica than that in D.

Fig. 5. Effects of quinclorac on ascorbate peroxidase (APX) activity in shoots of (a) Eleusine indica and (b) Digitaria adscendens after 12 h root treatment. NS: not significant (control vs. quinclorac treatment).

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Fig. 6. Effects of quinclorac on glutathione reductase (GR) activity in shoots of (a) Eleusine indica and (b) Digitaria adscendens after 12 h root treatment. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.01 (control vs. quinclorac treatment).

adscendens. Quinclorac (10 ␮M) did not enhance APX activity in the shoots of E. indica or D. adscendens (Fig. 5), although APX also plays a central role in scavenging H2 O2 to form water and oxygen to protect plants against oxidative stress (Vaidyanathan et al., 2003; Wei-Feng et al., 2008). More detailed studies of the activities of antioxidative enzymes in different compartments or in compartment-specific responses and quinclorac-induced ROS generation may be useful to clarify the mechanism of the herbicide-induced ROS production. The mean GR activities were 0.3 ± 0.0 and 0.1 ± 0.0 ␮mol NADPH oxidized g FW−1 min−1 in shoots of E. indica and D. adscendens 0 days after 12 h root treatment, respectively (Fig. 6a and b); the constitutive level of GR activity was 3.0-fold higher in E. indica than that in D. adscendens. In addition, 10 ␮M quinclorac did not affect GR activity in E. indica, but reduced the GR activity in D. adscendens 2 days after treatment. GR plays a key role in the protective system by converting oxidized glutathione to its reduced form (GSH) (Apel and Hirt, 2004). This activity increases the GSH/GSSG ratio, which is required for ascorbate regeneration (Azevedo Neto et al., 2005). In addition, it is known that increased GSH content could be associated with increased tolerance to oxidative stress (Meloni et al., 2003). In the present study, E. indica seedlings had higher constitutive levels of CAT, APX and GR activity than those of D. adscendens seedlings. In our previous study (Sunohara and Matsumoto, 2004), there were positive correlations between quinclorac sensitivity and the constitutive activities of antioxidative enzymes such as APX and GR in five grass species with different sensitivities to quinclorac: rice, maize, Echinochloa crus-galli Beauv. var. crus-galli, Echinochloa crus-galli var. formosensis Ohwi, and E. oryzicola Vasing. The present study indicated that E. indica has a better inherent enzymatic protection system against ROS than D. adscendens. The high antioxidative ability in E. indica could be one factor in quinclorac tolerance.

1.8 ± 0.0 ␮g leaf disc−1 and in D. ascendens, 0.3 ± 0.1 ␮g leaf disc−1 ; the chlorophyll content was 4.5-fold higher in E. indica than that in D. adscendens (Fig. 7a). In 500 mM H2 O2 -treated leaf discs, the mean chlorophyll contents were 1.7 ± 0.1 ␮g leaf disc−1 in E. indica and 0.3 ± 0.1 ␮g leaf disc−1 in D. adscendens; the chlorophyll content was 5.7-fold higher in E. indica than that in D. adscendens (Fig. 7a). These results indicate that better protection systems against ROS in E. indica alleviate the toxicity of H2 O2 more efficiently. The results further strengthen that the high antioxidant capacity could be involved in tolerance to quinclorac, which induces ROS overproduction. Quinclorac-induced phytotoxicity in several susceptible grasses has been considered to be due to the induction of ethylene precursor ACC-dependent cyanide (Grossmann and Kwiatkowski, 1995, 2000; Abdallah et al., 2006). Cyanide is known to be toxic to enzymes in many important metabolic pathways at and above 20 ␮M (Solomanson, 1981), and quinclorac-accumulated cyanide

3.5. Tolerance to H2 O2 toxicity Chlorophyll content was used as an index of sensitivity to ROS, because ROS induce decomposition of chlorophyll pigments. To investigate whether the high antioxidant capacity of E. indica contributed to overcoming ROS, leaf discs’ sensitivity to H2 O2 in E. indica and D. adscendens were compared. Fig. 7 shows the effects of H2 O2 treatment on chlorophyll content in leaf discs of E. indica and D. adscendens. Chlorophyll contents in leaf discs of non-treated controls of both species were similar, but they were higher in E. indica in 100, 200, 500, and 1000 mM H2 O2 -treated leaf discs (Fig. 7a and b). E. indica and D. ascendens were especially different in 200 and 500 mM H2 O2 -treated leaf discs. The mean chlorophyll content in 200 mM H2 O2 -treated leaf discs in E. indica was

Fig. 7. Effects of H2 O2 on chlorophyll content in leaf discs of Eleusine indica and Digitaria adscendens. Leaf discs of the plants were soaked in H2 O2 solution for 1 h in the dark, and then incubated in the solution at 25 ◦ C for 27 h under continuous light (4–7 ␮mol m−2 s−1 ). NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.01 (E. indica vs. D. adscendens).

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in shoot tissues of susceptible E. crus-galli (L.) P. Beauv. reached a maximum of 40 ␮M (Grossmann and Kwiatkowski, 1995). In addition, application of an ethylene biosynthesis inhibitor decreased quinclorac-induced phytotoxicity in susceptible grasses such as Echinochloa spp. and D. ischaemum (Lopez-Martinez et al., 1998; Abdallah et al., 2006). Therefore, accumulation of cyanide resulting from ethylene biosynthesis probably plays an important role in quinclorac-induced phytotoxicity in susceptible grasses. In our previous study (Sunohara et al., 2010), activation of ethylene biosynthesis by application of ACC, an intermediate of ethylene biosynthesis, reduced the growth of E. indica and D. adscendens. In addition, quinclorac (10 ␮M) did not induce growth reduction and ethylene production in E. indica but did so in D. adscendens seedlings. On the other hand, a representative synthetic auxin, 2,4D, rapidly induced huge ethylene production in both E. indica and D. adscendens seedlings, and the level of 2,4-D-induced ethylene in E. indica was much higher than that in D. adscendens (Sunohara et al., 2010). Therefore, failure of the pathway leading to the induction of ethylene biosynthesis by quinclorac may be an important factor for quinclorac tolerance in E. indica. However, quinclorac also induced oxidative injury in several susceptible grasses such as E. oryzicola Vasing. (Sunohara and Matsumoto, 2004), D. adscendens (Sunohara et al., 2010), and maize (Sunohara and Matsumoto, 2008). In the present study (1) quinclorac induced ROS overproduction in the roots of D. adscendens (Fig. 2b) (2) E. indica possessed better protection systems against ROS (Figs. 3–6), and 3) the toxicity of H2 O2 was alleviated in E. indica (Fig. 7). Therefore, the high antioxidant capacity in E. indica could be one factor underlying quinclorac tolerance. All the data presented above strongly suggest that quinclorac has additional oxidative actions accompanied by ROS overproduction in several susceptible grasses, and high quinclorac tolerance in E. indica may be a result of multiple factors such as the failure of activation of ethylene biosynthesis by quinclorac and high antioxidative capacity. Possible involvement of the capacity to metabolize cyanide in quinclorac tolerance in E. indica should be also considered in further studies. Acknowledgement This work was partially supported by Grant-in-Aid for Scientific Research (C) (No. 21580011) from the Japan Society for the Promotion of Science. References Abdallah, I., Fischer, A.J., Elmore, C.L., Saltveit, M.E., Zaki, M., 2006. Mechanism of resistance to quinclorac in smooth crabgrass (Digitaria ischaemum). Pest. Biochem. Physiol. 84, 38–48. Aebi, H.E., 1983. Catalase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 3. Verlag Chemie Weinheim, Deerfield Beach, FL, pp. 273–286. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Azevedo Neto, A.D., Prisco, J.T., Eneas-Filho, J., Medeiros, J.R., Gomes-Filho, E., 2005. Hydrogen peroxide pre-treatment induces salt stress acclimation in maize plants. J. Plant Physiol. 162, 1114–1122. Becker, L.B., Hoek, T.L.V., Shao, Z.H., Li, C.Q., Schumacker, P.T., 1999. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am. J. Physiol. Heart. Circ. Physiol. 277, 2240–2246. Berghaus, R., Wuerzer, B., 1987. The mode of action of the new experimental herbicide quinclorac (BAS 514 H). In: Proc. 11th Asian-Pac. Weed Sci. Soc. Conf., vol. 1 , pp. 81–87. Carter, W.O., Narayanan, P.K., Robinson, J.P., 1994. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol. 55, 253–258. Casano, L.M., Gomez, L.D., Lascano, H.R., Gonzalez, C.A., Trippi, V.S., 1997. Inactivation and degradation of CuZn-SOD by active oxygen species in wheat chloroplasts exposed photo oxidative stress. Plant Cell Physiol. 38, 433–440. Chappelle, E.W., Kim, M.S., McMurtrey III, J.E., 1992. Ratio analysis of reflectance spectra (RARS): an algorithm for the remote estimation of the concentrations of chlorophyll a, chlorophyll b, and carotenoids in soybean leaves. Remote Sens. Environ. 39, 239–247.

Chism, W.J., Bingham, S.W., Shaver, R.L., 1991. Uptake, translocation and metabolism of quinclorac in two grass species. Weed Technol. 5, 771–775. Davies, K.J.A., 1987. Protein damage and degradation by oxygen radicals. J. Biol. Chem. 262, 9895–9901. Foyer, C.H., Lopez-Delgado, H., Dat, J.F., Scott, I.M., 1997. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signaling. Physiol. Plant 100, 241–254. Fridovich, I., 1986. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247, 1–11. Grant, J.J., Loake, G.J., 2000. Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol. 124, 21–29. Grossmann, K., 1998. Quinclorac belongs to a new class of highly selective auxin herbicides. Weed Sci. 46, 707–716. Grossmann, K., Kwiatkowski, J., 1993. Selective induction or ethylene and cyanide biosynthesis appears to be involved in the selective of the herbicide quinclorac between rice and barnyard grass. J. Plant Physiol. 142, 457–465. Grossmann, K., Kwiatkowski, J., 1995. Evidence for a causative role of cyanide, derived from ethylene biosynthesis, in the herbicidal mode of action of quinclorac in barnyard grass. Pest. Biochem. Physiol. 51, 150–160. Grossmann, K., Kwiatkowski, J., 2000. The mechanism of quinclorac selectivity in grasses. Pest. Biochem. Physiol. 66, 83–91. Grossmann, K., Scheltrup, F., 1997. Selective induction of 1-aminocyclopropane-1carboxylate acid (ACC) synthase activity is involved in the selectivity of the auxin herbicide quinclorac between barnyard grass and rice. Pest. Biochem. Physiol. 58, 145–153. Gupta, A.S., Heinen, J.L., Holaday, A.S., Burke, J., Allen, R.D., 1993. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 90, 1629–1633. Halliwell, B., Foyer, C.H., 1978. Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography. Planta 139, 9–17. Imlay, J.A., Linn, S., 1988. DNA damage and oxygen radical toxicity. Science 240, 1302–1309. Koo, S.J., Kwon, Y.W., Cho, K.Y., 1991. Differences in herbicidal activity, phytotoxic symptoms and auxin activity of quinclorac among plant species compared with 2,4-D. Weed Res. Jpn. 36, 311–317. Koo, S.J., Neal, J.C., DiTomaso, J.M., 1996. 3,7-dichloroquinolinecarboxylic acid inhibits cell-wall biosynthesis in maize roots. Plant Physiol. 112, 1383–1389. Lopez-Martinez, N., Shimabukuro, R.H., Prado, R.D., 1998. Effect of quinclorac on auxin-induced growth, transmembrane proton gradient and ethylene biosynthesis in Echinochloa spp. Aust. J. Plant Physiol. 25, 851–857. Meloni, D.A., Oliva, M.A., Martinez, C.A., Cambraia, J., 2003. Photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ. Exp. Bot. 49, 69–76. Menck, B.H., Rosebrock, H., Unglaub, W., Kibler, E., 1985. BAS514. H-quinclorac field experience to control Echinochloa crus-galli in rice. In: Proc. 10th Asian-Pac. Weed Sci. Soc. Conf., vol. 1 , pp. 107–113. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Nakano, Y., Asada, K., 1987. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 28, 131–140. Nagata, T., Todoriki, S., Masumizu, T., Suda, I., Furuta, S., Du, Z., Kikuchi, S., 2003. Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis. J. Agric. Food Chem. 51, 2992–2999. Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D., Hancock, J.T., 2002. Hydrogen peroxide and nitric oxide as signaling molecules in plants. J. Exp. Bot. 53, 1237–1247. Niki, E., 1991. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54, 1119–1124. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: Keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. 49, 249–279. Ohta, Y., Yamamoto, K., Deguchi, M., 1970. Effect of calcium supply and plant age on the distribution of calcium salts in rice. Jpn. J. Soil Sci. Plant Nutr. 41, 19–26 (in Japanese). Rosen, G.M., Freeman, B.A., 1984. Detection of superoxide generated by endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 81, 7269–7273. Scandalios, J.G., Guan, L., Polidoros, A.N., 1997. Catalases in plants: gene structure, properties, regulation and expression. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 343–406. Schabenberger, O., Tharp, B., Kells, J., Penner, D., 1999. Statistical tests for hormesis and effective dosages in herbicide dose-response. Agronomy J. 91, 713–721. Seefeldt, S.S., Jensen, J.E., Fuerst, E.P., 1995. Log–logistic analysis of herbicide doseresponse relationships. Weed Technol. 9, 218–227. Solomanson, L.P., 1981. Cyanide as a metabolic inhibitor. In: Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J., Wissing, F. (Eds.), Cyanide in Biology. Academic Press, London, pp. 11–28. Sunohara, Y., Kobayashi, M., Matsumoto, H., 2003a. Light effect on induction of 1aminocyclopropane-1-carboxylic acid synthase activity in quinclorac- or 2,4-Dtreated maize seedlings. In: Proc. 19th Asian-Pac. Weed Sci. Soc. Conf. II , pp. 828–836. Sunohara, Y., Kobayashi, M., Matsumoto, H., 2003b. Light induction of 1aminocyclopropane-1-carboxylic acid synthase activity in quinclorac-treated maize seedlings. J. Pest. Sci. 28, 18–23.

Y. Sunohara et al. / Environmental and Experimental Botany 74 (2011) 74–81 Sunohara, Y., Matsumoto, H., 1997. Comparative physiological effects of quinclorac and auxins, and light involvement in quinclorac-induced chlorosis in corn leaves. Pest. Biochem. Physiol. 58, 125–132. Sunohara, Y., Matsumoto, H., 2004. Oxidative injury induced by the herbicide quinclorac on Echinochloa oryzicola Vasing. and the involvement of antioxidative ability in its highly selective action in grass species. Plant Sci. 167, 597–606. Sunohara, Y., Matsumoto, H., 2008. Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue. Phytochemistry 69, 2312–2319. Sunohara, Y., Shirai, S., Wongkantrakorn, N., Matsumoto, H., 2010. Sensitivity and physiological responses of Eleusine indica and Digitaria adscendens to herbicide quinclorac and 2, 4-D. Environ. Exp. Bot. 68, 157–164. Takematsu, T., Ichizen, N., 1997. Monocotyledoneae. Weeds of the World, vol. 3. Zenkoku Noson Kyoiku Kyokai, Tokyo (in Japanese). Tresch, S., Grossmann, K., 2003a. Quinclorac does not inhibit cellulose (cell wall) biosynthesis in sensitive barnyard grass and maize roots. Pest. Biochem. Physiol. 75, 73–78. Tresch, S., Grossmann, K., 2003b. Erratum to “Quinclorac does not inhibit cellulose (cell wall) biosynthesis in sensitive barnyard grass and maize roots”. Pest. Biochem. Physiol. 76, 70–71.

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Vaidyanathan, H., Sivakumar, P., Chakrabarty, R., Thomas, G., 2003. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)-differential response in salt-tolerant and sensitive varieties. Plant Sci. 165, 1411–1418. Vranová, E., Inzé, D., van Breusegem, F., 2002. Signal transduction during oxidative stress. J. Exp. Bot. 53, 1227–1236. Wei-Feng, X., Wei-Ming, S., Ueda, A., Takabe, T., 2008. Mechanisms of salt tolerance in transgenic Arabidopsis thaliana carrying a peroxisomal ascorbate peroxidase gene from barley. Pedosphere 18, 486–495. Wuerzer, B., Berghaus, R., 1985. Substituted quinoline carboxylic acid-new elements in herbicide systems. In: Proc. 10th Asian-Pac. Weed Sci. Soc. Conf., vol. 1 , pp. 177–184. Yamamoto, Y., Kobayashi, Y., Devi, S.R., Rikiishi, S., Matsumoto, H., 2002. Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells. Plant Physiol. 128, 63–72. Zawierucha, J.E., Penner, D., 2000. Absorption, translocation, metabolism, and spray retention of quinclorac in Digitaria sanguinalis and Eleusine indica. Weed Sci. 48, 296–301.