The Mechanism of Quinclorac Selectivity in Grasses

Pesticide Biochemistry and Physiology 66, 83–91 (2000) doi:10.1006/pest.1999.2461, available online at http://www.idealibrary.com on

The Mechanism of Quinclorac Selectivity in Grasses Klaus Grossmann1 and Jacek Kwiatkowski BASF Agricultural Center Limburgerhof, D-67114 Limburgerhof, Germany Received April 16, 1999; accepted October 15, 1999 The mechanism of selectivity of the auxin herbicide quinclorac was studied in susceptible and resistant biotypes of Echinochloa hispidula and E. crus-galli; in grass species of different susceptibility like Brachiaria platyphylla, Setaria viridis, E. colonum, and Digitaria sanguinalis; and in tolerant crop rice (Oryza sativa cv. Thaibonnet). No significant differences in uptake, translocation, or metabolism of quinclorac between resistant and sensitive grasses were found. Hence, the mechanism of selectivity is target-site-based. It has been demonstrated that cyanide, which accumulates as a coproduct during stimulation of ethylene biosynthesis, is the primary phytotoxic principle in the herbicidal mode of action of quinclorac in E. crus-galli (K. Grossmann and J. Kwiatkowski, 1995, Pestic. Biochem. Physiol. 51, 150). Following root treatment of plants at the third leaf stage with 100 mM quinclorac in hydroponics, the reductions in shoot growth of plants were closely correlated with the stimulation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity and with subsequent increases in endogenous concentrations of ACC and cyanide. When compared quinclorac with other auxin herbicides including quinmerac, a-naphthaleneacetic acid and dicamba, lower ACC and cyanide levels and correlated lower growth effects were found after treatment of D. sanguinalis. In quinclorac-resistant grass species and biotypes, ACC synthase activity was not induced and plants exhibited no significant changes in ACC and cyanide levels. Thus, the selective induction of ACC synthase activity, which ultimately leads to cyanide accumulation in the tissues of susceptible plants, plays the primary role for the selectivity of grasses toward quinclorac. This selectivity could be at the level of quinclorac perception or subsequent signal transduction via the auxin pathway leading to regulation of gene expression or posttranscriptional de novo enzyme synthesis. In rice, tolerance to quinclorac could be additionally favored by a higher b-cyanoalanine synthase activity, the main HCN-detoxifying enzyme. q 2000 Academic Press

INTRODUCTION

United States and the Far East) and E. hispidula (barnyard grass, in Southern Europe), are also of importance (4, 5). Quinclorac controls a broad range of the about 50 Echinochloa species and subspecies globally known (4–6). However, some of the numerous Echinochloa species or biotypes in Southern Europe and the United States are less sensitive to quinclorac (4). This has been found to be independent of a history of quinclorac application (4). In Spain, biotypes of E. crus-galli and E. hispidula, with reduced sensitivity, and a resistant biotype of E. hispidula have been described (4, 7, 8). The precise mechanism of resistance of these biotypes is not known. Studies suggested that it is not based on differences in uptake, translocation, and metabolism of the herbicide (4, 7). Quinclorac is a systemic herbicide which is readily absorbed by germinating seeds, roots, and leaves and is translocated in the plant both

The quinolinecarboxylic acid quinclorac (3,7dichloro-8-quinolinecarboxylic acid, BAS 514H, Facet) belongs to a new class of highly selective, auxin herbicides (1–3). Quinclorac is used in rice and has also been developed for application in turfgrass areas, spring wheat, and chemical fallow. The herbicide effectively controls important dicot and monocot weeds, including grass species of Echinochloa, Digitaria, and Setaria, with excellent crop safety. In rice cultivation, the barnyard grass (Echinochloa crusgalli) is the major grass weed worldwide. Regionally, other Echinochloa species, such as E. colonum (jungle rice, in South America, the 1 To whom correspondence should be addressed at BASF Agricultural Center Limburgerhof, P.O. Box 120, D-67114 Limburgerhof, Germany. Fax: 149 621 60 271 76. E-mail: [email protected].

83 0048-3575/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

84

GROSSMANN AND KWIATKOWSKI

acropetally and basipetally (3, 9, 10). In barnyard grass, Digitaria sanguinalis (large crabgrass), Eleusine indica (goosegrass), and rice, quinclorac was metabolized at a moderate rate (3, 7, 9, 11). After 24 h, 5–10% of the absorbed quinclorac was transformed into a polar metabolite. No qualitative or quantitative differences between metabolism in root and shoot tissue were observed (3, 9, 12). Herbicidal effects in barnyard grass are characterized by growth inhibition, particularly of the shoot, which remained stunted and showed progressive chlorosis, first on the growing areas of the youngest leaf blades, followed by wilting and necrosis. The primary mode of action of quinclorac affects the phytohormonal system in sensitive plants by mimiking an auxin overdose (3). Species-selective inhibition of cell wall biosynthesis was also suggested to be involved (13). Recent investigations have shown that in barnyard grass the phytotoxic effects of quinclorac are caused through the accumulation, particularly in the shoot tissue, of cyanide, which ultimately derives from herbicide-stimulated 1-aminocyclopropane-1-carboxylic acid (ACC)2 synthase activity in ethylene biosynthesis (1, 3, 7, 8, 12, 14). Specific interference with the induction process, leading to de novo synthesis of ACC synthase within the first hours after treatment, has been suggested as the mechanism of action of quinclorac (3, 12). Consequently, the production of ACC increases and phytotoxic cyanide subsequently accumulates as a coproduct of increased ethylene synthesis during the oxidation of ACC catalyzed by ACC oxidase (8, 15). This has been shown to be a common effect of auxin herbicides in sensitive grasses (3). Corresponding biochemical changes and growth effects were not observed in resistant rice (8, 12, 14). Since no significant differences in uptake, translocation, or metabolism of quinclorac between rice and barnyard grass could be found, a target-site-based mechanism of selectivity has been suggested (7, 8, 12–14). In the present communication the mechanism of resistance to quinclorac was elucidated in 2 Abbreviations used: ACC, 1-aminocyclopropane-1carboxylic acid.

biotypes of E. hispidula (R) found in Spain and E. crus-galli (R) found in the United States. The uptake, translocation, and metabolism of the herbicide, the effects on ACC synthase activity, and the levels of ACC were studied in plants treated hydroponically within the most important first 7 h of herbicide exposure (1, 12). In addition, the changes in shoot and root fresh weight and the endogenous levels of ACC and cyanide were studied 75 h after treatment. The effects were directly compared with those (i) in a susceptible biotype of E. hispidula (S) from Korea and (ii) in differentially sensitive Echinochloa species, including E. colonum from the United States and E. crus-galli from Spain. In order to investigate whether quinclorac-induced accumulation of cyanide derived from stimulated ACC synthesis is also the phytotoxic principle in other susceptible grasses, the response of D. sanguinalis, Setaria viridis (green foxtail), and Brachiaria platyphylla (broadleaf signalgrass) were tested. Additionally, the tolerant crop rice (Oryza sativa) was included in this investigation. Furthermore, the basal activity of the b-cyanoalanine synthase, which detoxifies cyanide in plant tissue (15), was determined. Our investigations also approached the question of whether cyanide accumulation derived from increased ACC synthesis and the consequent growth inhibition is a common effect of auxintype herbicides. Thus, several representatives of the known chemical classes including dicamba, a-naphthaleneacetic acid, quinmerac, and quinclorac were examined in D. sanguinalis. MATERIALS AND METHODS

Chemicals The herbicide 3,7-dichloro-8-quinolinecarboxylic acid (quinclorac, BAS 514H) and [14C]quinclorac (.99% radiochemical purity, sp act 362 MBq/mmol, 1.5 MBq/mg, labeled at C2 atom in the heterocycle) from BASF Aktiengesellschaft, Ludwigshafen, Germany, was used. The other auxin herbicides 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 7-chloro-3methyl-quinoline-8-carboxylic acid (quinmerac), and a-naphthaleneacetic acid were from

MECHANISM OF QUINCLORAC SELECTIVITY IN GRASSES

Riedel-de Haen (Seelze, Germany) and BASF Aktiengesellschaft. ACC was obtained from Calbiochem (Frankfurt, Germany). Cultivation of Plants in Hydroponics Seedlings of E. hispidula L., a resistant (R) biotype, BT 63 B, from Spain, and a susceptible (S) biotype from Korea, BT 26 C; E. crus-galli (L.) P. Beauv., a resistant (R) biotype collected in the United States (Mississippi area) and a susceptible (S) biotype from Spain (seed material was kindly supplied by Dr. O. Schmidt, U. Hayler, and L. J. Newsom, BASF Agricultural Center Limburgerhof, Germany); E. colonum (L.) Link; D. sanguinalis (L.) Scop; S. viridis (L.) P. Beauv; B. platyphylla (Griseb.) Nash; and O. sativa L. cv. Thaibonnet were raised to the third leaf stage and transferred to 320-ml glass vessels containing Linsmaier–Skoog medium (30 plants per vessel, 10 replicates; 16/8-h light/ dark cycles at 25/228C, 75% relative humidity) as previously described (1). Quinclorac and the other auxin herbicides were added in acetone solution and the organic solvent was allowed to volatilize before the vessels were loaded with medium and plants. At various times after treatment, the shoots and roots from parallel vessels were harvested, immediately frozen in solid CO2, and stored at 2808C. All experiments were repeated at least twice and proved to be reproducible. The results of a representative experiment are shown. Determination of ACC, ACC Synthase Activity, and Tissue HCN and b-Cyanoalanine Synthase Activity Plant material was powdered under liquid nitrogen and samples (100 mg; three replicates) were extracted with 5 ml 70% aqueous ethanol for determination of ACC. After centrifugation, the supernatant was passed through a C18reversed-phase prepacked column (Seppak, Waters, Ko¨nigstein, Germany) for purification. A 3-ml aliquot of the effluent was concentrated under vacuum to dryness and brought to a volume of 2.75 ml with 2 ml double-distilled water

85

and 0.75 ml 2 M KOH solution. The ACC content was assayed by converting it directly to ethylene with NaOCl in the presence of HgCl2 as described elsewhere (12, 16). Ethylene was measured by gas chromatography (16). ACC synthase (EC 4.4.1.4) was extracted and assayed as described (17). After passing through a Sephadex G-25 column, the extract from powdered plant material was used in the ACC synthase assay mixture. After incubation in the presence of S-adenosylmethionine, the ACC produced was determined by chemical conversion to ethylene (16). Background levels of ACC, which were analyzed in samples in which the reaction was stopped at time zero, were subtracted. All assays were performed in triplicate. Tissue HCN was determined using a modification of the method described previously (1). A sample of powdered plant material (1.5 g) was transferred to a glass vial (25 mm in diameter, 38 mm in height), the lid of which contained a filter paper disk to which 350 ml 1.5 M NaOH had been applied. Sealing the vial with the lid positioned the filter over the plant material. Then, 2.5 ml 5% H2SO4 was injected into the sample through a perforation in the side of the vial, which was subsequently sealed. The resultant acidified brei was stirred at 228C for 5 h to allow evolved HCN to be trapped by the filter. The filter was then eluted with NaOH for 1 h and the cyanide content was determined using the colorimetric method developed by Lambert et al. (18). The activity of b-cyanoalanine synthase (EC 4.4.1.9) was extracted from powdered shoot material and assayed in the presence of sodium cyanide and L-cysteine as the substrates according to (1, 14). The enzyme activity was determined colorimetrically after conversion of released H2S (from cysteine) to methylene blue (in the presence of N, N-dimethyl-p-phenylenediamine and ferric chloride) using Na2S as the standard. Uptake, Translocation, and Metabolism of [14C] Quinclorac Plants at the third leaf stage were placed in plastic vials (25 mm in diameter, 38 mm in height; Greiner, Nu¨rtingen, Germany) containing 10 ml of 1/2 strength Linsmaier-Skoog

86

GROSSMANN AND KWIATKOWSKI

medium (19) and 100 mM [14C]quinclorac (37 KBq/vial). The vials were closed with plastic covers with slits through which the plants were fitted upright (four plants/vial). Subsequently, the vials (10 replicates) were placed in growth chambers (continuous light, 530 mmol m22 s21 photon irradiance, 258C). After 7 h, plants were removed from the medium and the roots were carefully washed. Then, plants were sectioned into shoot and root, and fresh weights were determined. Plant parts were dried at 408C for 3 days and combusted in a biological materials oxidizer (Oxymat OX 300, Zinsser, Frankfurt, Germany). The evolved 14CO2 was absorbed in Oxosolve C-400 LS cocktail (Zinsser) and quantified by scintillation counting. The concentration of the compound per unit fresh weight was calculated on the basis of the specific activity of the [14C]quinclorac (1.5 KBq mg21). For study of the metabolism of [14C]quinclorac, shoot and root material from parallel vials (10 replicates) was harvested, immediately frozen in solid CO2, and stored at 2808C. Plant material (approx. 1 g) was homogenized in 5 ml 100% methanol with an Ultra-Turrax (IKA, Staufen, Germany) and extracted three times for 30 min at 48C. After centrifugation, the supernatants were combined, concentrated to dryness by rotary evaporation, redissolved in double-distilled water (3 ml), and extracted three times with 3 ml n-hexane. The aqueous extract was acidified with 1 M HCl to pH 2 and extracted three times with 3 ml ethyl acetate. The combined ethyl acetate phases were concentrated to dryness and redissolved in 200 ml 100% methanol. Then, 20 ml of the sample was separated by reverse-phase HPLC on a Spherisorb C18-2, 5-mm column (250 3 4 mm, Macherey-Nagel, Du¨ren, Germany) using a linear gradient from 20% acetonitrile in water to 100% acetonitrile. The gradient sweep time was 20 min at a flow rate of 1 ml min21. Radioactivity of the fractions containing quinclorac (10.7 min retention time) and unidentified metabolites (retention time 9.0 and 9.9 min) was determined using a HPLC-coupled radioactivity monitor (LB 506 C, Berthold, Wildbad, Germany). Recovery of absorbed radioactivity was above 70% in all cases after the extraction and HPLC procedure.

RESULTS

Effects on Ethylene Biosynthesis, Levels of Tissue Cyanide, and ß-Cyanoalanine Synthase Activity and Growth In barnyard grass (E. crus-galli), quinclorac has been shown to stimulate the induction of ACC synthase activity which leads, particularly in the shoot tissue, to an accumulation of phytotoxic concentrations of cyanide, formed as a coproduct during increased ACC and ethylene synthesis (1, 12, 14, 15). After root treatment with 100 mM quinclorac for 75 h, shoot fresh weight was reduced in E. hispidula (R), O. sativa, E. crus-galli (R), B. platyphylla, E. crusgalli (S), S. viridis, E. colonum, E. hispidula (S), and D. sanguinalis by 11, 21, 25, 60, 69, 73, 76, 88, and 92%, respectively (Figs. 1–3). Root growth was affected approximately in the same order, but to a lesser extent (Figs. 1 and 2). Reductions in the shoot fresh weight of plants closely correlated with increased ACC and particularly cyanide levels in the shoot tissues (Figs. 1–3). The values were referred to shoot dry weight, since the fresh to dry weight ratio in the susceptible species decreased 75 h after quinclorac treatment (data not shown). Susceptible species, such as D. sanguinalis and E. hispidula (S), responded to treatment with an increase in ACC and cyanide levels in the shoot tissue of up to 170- and 9-fold, respectively, relative to controls (Fig. 1). In accordance with results obtained in E. crus-galli (1), in D. sanguinalis exposed to quinclorac, the time course of cyanide accumulation correlated with the reduction in the shoot fresh weight, i.e., the herbicidal effect (Fig. 4). In contrast, resistant species or biotypes, such as E. hispidula (R), E. crus-galli (R), and O. sativa, showed only slight changes in cyanide and ACC levels and plant growth (Figs. 1 and 2). Within 7 h, the enzymatic activity of ACC synthase, immediately preceding ethylene and cyanide production, was stimulated by quinclorac in the shoot tissues of the different grass species and biotypes according to their herbicidal sensitivity. At this time, no changes in growth parameters were observed (data not shown). Concomitantly, ACC levels in shoot and root were changed selectively. In susceptible

MECHANISM OF QUINCLORAC SELECTIVITY IN GRASSES

87

FIG. 1. Effects of quinclorac on shoot and root growth (reduction of fresh weight), ACC synthase activity, and ACC and cyanide levels in a susceptible (S) and a resistant (R) biotype of Echinochloa hispidula (EH) and E. crus-galli (EG). Plants at third leaf stage were root treated with 100 mM quinclorac hydroponically. The activity of ACC synthase in shoot tissue and ACC contents in shoots and roots were determined after 7 h of treatment. For determination of changes in shoot and root fresh weight and ACC and cyanide levels in shoot tissue, plants were treated with quinclorac for 75 h. Vertical bars represent SE of the mean. Control values 6 SE representing 0% change of shoot fresh weight (mg/shoot), 86 6 13 (S-EH), 56 6 4 (R-EH), 49 6 2 (SEG), 90 6 10 (R-EG); root fresh weight (mg/root), 43 6 6 (S-EH), 26 6 2 (R-EH), 29 6 1 (S-EG), 44 6 5 (R-EG); ACC synthase activity (pmol/g shoot fresh wt ? h), 4 6 2 (S-EH), 3 6 0 (R-EH), 27 6 1 (S-EG), 7 6 2 (R-EG); root ACC content (nmol/g fresh wt, 7 h). 0.54 6 0.04 (S-EH), 0.87 6 0.03 (R-EH), 2.80 6 0.24 (S-EG), 2.56 6 0.24 (R-EG); shoot ACC content (nmol/g FW, 7 h), 0.42 6 0.07 (S-EH), 0.64 6 0.07 (R-EH), 0.97 6 0.08 (S-EG), 1.30 6 0.39 (R-EG); shoot ACC content (nmol/g DW, 75 h), 2.6 6 0.3 (S-EH), 7.6 6 0.4 (R-EH), 4.9 6 0.1 (S-EG), 12.0 6 0.6 (R-EG); cyanide content (nmol/g DW), 36.0 6 3.6 (S-EH), 68.4 6 3.6 (R-EH), 92.6 6 11.4 (S-EG), 135.8 6 11.2 (R-EG).

species, such as D. sanguinalis and E. hispidula (S), root treatment with 100 mM quinclorac for 7 h increased ACC synthase activity and ACC levels in the shoot tissue up to 8- and 12-fold, respectively, relative to the control (Fig. 1). In the resistant species O. sativa, E. hispidula (R), and E. crus-galli (R), quinclorac did not stimulate ACC synthase activity and ACC levels (Figs. 1 and 2). In order to evaluate whether ACC and cyanide accumulation and the consequent growth response is a common effect of auxin herbicides

in susceptible grass species, several representatives of the known chemical classes of auxin herbicides were examined in D. sanguinalis (Figs. 5A and 5B). When compared with quinclorac and based on shoot dry weight, lower ACC (Fig. 5A) and cyanide levels (Fig. 5B) in the shoot tissue and correlated lower reductions in shoot fresh weight were found after exposure to quinmerac, a-naphthaleneacetic acid, and dicamba. Exogenous applied KCN mimicked effects on shoot growth at comparable endogenous cyanide concentrations (Fig. 5B).

88

GROSSMANN AND KWIATKOWSKI

FIG. 2. Effects of quinclorac on shoot and root growth (reduction of fresh weight), ACC synthase activity, and ACC and cyanide levels in Echinochloa colonum (EC), Digitaria sanguinalis (DS), Setaria viridis (SV), and Oryza sativa (OS). Plants at the third leaf stage were root treated with 100 mM quinclorac hydroponically. The activity of ACC synthase in shoot tissue and ACC contents in shoots and roots were determined after 7 h of treatment. For determination of changes in shoot and root fresh weight and ACC and cyanide levels in shoot tissue, plants were treated with quinclorac for 75 h. Vertical bars represent SE of the mean. Control values 6 SE representing 0% change of shoot fresh weight (mg/shoot), 105 6 9 (EC), 354 6 38 (DS), 369 6 28 (SV), 67 6 8 (OS); root fresh weight (mg/root), 54 6 7 (EC), 132 6 11 (DS), 130 6 9 (SV), 76 6 1 (OS); ACC synthase activity (pmol/g shoot fresh wt ? h), 10 6 2 (EC), 7 6 1 (DS), 33 6 1 (SV), 38 6 2 (OS); root ACC content (nmol/g fresh wt, 7 h), 1.33 6 0.13 (EC), 1.21 6 0.08 (DS), 1.83 6 0.03 (SV), 1.74 6 0.11 (OS); shoot ACC content (nmol/g fresh wt, 7 h), 0.50 6 0.04 (EC), 0.58 6 0 (DS), 1.23 6 0.05 (SV), 1.02 6 0.06 (OS); shoot ACC content (nmol/g dry wt, 75 h), 6.6 6 1.7 (EC), 4.8 6 0.2 (DS), 6.3 6 0.1 (SV), 9.5 6 1.6 (OS); cyanide content (nmol/g dry wt), 41.6 6 4.2 (EC), 47.1 6 8.6 (DS), 19.0 6 2.7 (SV), 61.7 6 4.0 (OS).

As an additional mechanism of selectivity, the enzymatic activity of the b-cyanoalanine synthase, which detoxifies HCN in plant tissue (15), could be a consideration (14). In untreated shoot tissue, higher values of ß-cyanoalanine synthase activity could be detected in O. sativa (1012 nmol g fresh wt21 h21), compared with E. crusgalli (167 nmol g21 h21), D. sanguinalis (461 nmol g21 h21), and S. viridis (551 nmol g21 h21). In O. sativa, this is in accordance with lower endogenous cyanide levels and with a higher tolerance of plants to damage by KCN application (14).

Absorption, Translocation, and Metabolism of [14C]Quinclorac After treatment of plants at the third leaf stage with 100 mM [14C]quinclorac for 7 h in hydroponics, root uptake and translocation of radioactivity were studied in O. sativa, D. sanguinalis, S. viridis, E. colonum, and resistant (R) and susceptible (S) biotypes of E. hispidula and E. crusgalli. Based on plant fresh weight, root uptake of quinclorac in the Echinochloa species and biotypes and in Digitaria was in a similar range which was approximately 1.7-fold higher than

MECHANISM OF QUINCLORAC SELECTIVITY IN GRASSES

89

that in Setaria and rice (Table 1). The amounts of C compound found in the shoot tissue reached similar values in the (R) compared to those in the (S) biotypes of E. hispidula and E. crusgalli, respectively, after 7 h of treatment (Table 1). Compared to E. hispidula, the other grass species partitioned similar but lower amounts of 14 C compound into the shoot (Table 1). In all grass species and biotypes, quinclorac was metabolized at only a moderate rate (Table 1). After root treatment with 100 mM [14C]quinclorac for 7 h, no qualitative or significant quantitative differences between metabolism in the various grass species and the biotypes were observed (Table 1). Approximately 90% of the absorbed radioactivity was found as unchanged herbicide in the shoot and root tissues (Table 1). In conclusion, the selective action of quinclorac is not influenced by differential metabolism and only slightly by differences in uptake and translocation. 14

FIG. 3. Correlation between the herbicide action of quinclorac (reduction of shoot fresh weight) and its effect on cyanide contents in shoots of different grass species and biotypes. Plants of Brachiaria platyphylla (BP), Digitaria sanguinalis (DS), a susceptible (S) and a resistant (R) biotype of Echinochloa hispidula (EH), and E. crus-galli (EG), E. colonum (EC), Oryza sativa (OS), and Setaria viridis (SV) at the third leaf stage were root treated with 100 mM quinclorac hydroponically for 75 h and shoot fresh weight and cyanide contents in shoots were determined. Control values 6 SE representing 100% of shoot fresh weight (mg/shoot), 60 6 4 (BP); cyanide content (nmol/g dry wt), 146.2 6 13.6 (BP). The other data are depicted from Figs. 1 and 2.

FIG. 4. Time course of the effects of 100 mM quinclorac on shoot growth and the content of cyanide in shoot tissue of root-treated Digitaria sanguinalis plants at the second leaf stage. Control values 6 SE, 72 h after incubation, representing 100% of shoot fresh weight (mg/shoot), 185 6 22; cyanide content (nmol/g dry wt), 115 6 5.

DISCUSSION

In the quinclorac-resistant and -susceptible grass species and biotypes studied, close correlations were demonstrated between their sensitivity to the herbicide and the endogenous levels of cyanide, ACC, and ACC synthase activity. Similarly, stimulation of ethylene formation by quinclorac positively correlated with the extent of the phytotoxic effects (3, 7, 8, 13). In contrast, no significant differences in uptake, translocation, or metabolism of quinclorac between resistant and sensitive grasses were found (Table 1; 3, 7, 9, 11, 12). Hence, a target-site-based mechanism of selectivity is suggested. It is concluded that the induction of ACC synthase activity, which ultimately leads to increased cyanide accumulation, plays the primary role. As shown in D. sanguinalis, this appears to be an effect common to auxin herbicides in susceptible grasses. A selective perception or signal transduction of quinclorac via the auxin pathway leading to increased gene expression or posttranscriptional regulation of de novo enzyme synthesis of ACC synthase is hypothesized. In resistant species or biotypes, corresponding processes appear to be not elicited by quinclorac. In vivo

90

GROSSMANN AND KWIATKOWSKI

FIG. 5. Correlation between the effects of selected auxin herbicides (dicamba, DC; a-naphthaleneacetic acid, NAA; quinclorac, QC; quinmerac, QM) applied at 100 mM and KCN (applied at 800 mM) on fresh weight and ACC (A) and cyanide content (B) in shoots of Digitaria sanguinalis. Plants at the second leaf stage were root treated for 75 h. Control values 6 SE representing 100% of shoot fresh weight (mg/shoot), 138 6 24; ACC content (nmol/g dry wt), 4.8 6 0.2; cyanide content (nmol/g dry wt), 109 6 12.

experiments with suspension-cultured soybean and parsley cells demonstrated that quinclorac and quinmerac activate phospholipase A, a putative component in auxin-induced signal transduction (20), to a similar level as 2-methyl-4chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, and indole3-propionic acid (R. Paul and G. Scherer, University of Hannover, Germany; personal communication). Possibly, selective interference

with a component such as this in auxin signaling might be involved in the selectivity of quinclorac in grasses. In rice, tolerance to quinclorac could be additionally increased by a higher b-cyanoalanine synthase activity. ACKNOWLEDGMENTS We thank Dr. O. Schmidt, U. Hayler, and L. J. Newsom for providing seed material of Echinochloa species and biotypes; Professor Dr. D. Penner, Michigan State University, East

TABLE 1 Root Absorption, Translocation, and Metabolism of [14C]Quinclorac in Grassesa Metabolism radioactivity [cpm (% of total)] Root absorption and translocation (mg 14C compound/g fresh wt) Plant species E. hispidula (S) E. hispidula (R) E. crus-galli (S) E. crus-galli (R) E. colonum D. sanguinalis S. viridis O. sativa a

Root 20.4 15.8 14.8 13.3 14.4 16.8 9.6 9.1

6 6 6 6 6 6 6 6

3.1 0.8 0.6 1.1 2.0 0.7 0.5 0.3

Shoot 8.5 7.4 3.9 3.4 3.7 4.9 3.5 5.8

6 6 6 6 6 6 6 6

0.8 0.6 0.4 0.5 0.5 0.3 0.2 0.6

Root

Quinclorac 63,696 40,608 69,678 39,550 45,579 39,304 18,288 78,688

(92) (87) (96) (83) (87) (95) (94) (94)

Metabolites 5264 5848 2964 8273 6793 2016 1096 4640

(8) (13) (4) (17) (13) (5) (6) (6)

Shoot Total extract separated 68,960 46,456 72,642 47,823 52,372 41,320 19,384 83,328

(100) (100) (100) (100) (100) (100) (100) (100)

Quinclorac 44,080 29,280 25,926 23,763 38,445 42,041 34,784 23,224

(90) (91) (93) (85) (92) (91) (92) (96)

Plants at the third leaf stage were root treated with 100 mM of [14C]quinclorac for 7 h.

Metabolites 4240 2784 1978 4226 3572 4083 3000 1072

(10) (9) (7) (15) (8) (9) (8) (4)

Total extract separated 48,840 32,064 27,904 27,989 42,017 46,124 37,784 24,296

(100) (100) (100) (100) (100) (100) (100) (100)

MECHANISM OF QUINCLORAC SELECTIVITY IN GRASSES Lansing, Michigan, and Dr. J. Zawierucha for valuable discussion; and Mr. A. Akers for critical reading of the English manuscript. 10.

REFERENCES 1. K. Grossmann and J. Kwiatkowski, Evidence for a causative role of cyanide, derived from ethylene biosynthesis, in the herbicidal mode of action of quinclorac in barnyard grass, Pestic. Biochem. Physiol. 51, 150 (1995). 2. B. Wuerzer and R. Berghaus, Substituted quinolinecarboxylic acids—New elements in herbicide systems, Proc. 10th Asian-Pac. Weed Sci. Soc. Conf. 177 (1985). 3. K. Grossmann, Quinclorac belongs to a new class of highly selective auxin herbicides, Weed Sci. 46, 707 (1998). 4. N. Lopez-Martinez, R. De Prado, W. Rademacher, H. Walter, G. Marshall, and O. Schmidt, Differential response of Echinochloa species and biotypes to quinclorac, in “Resistance 97, Abstract of the International Conference at IACR-Rothamsted,” Harpenden, Herts, UK, 1997. 5. O. Schmidt, O. Aurich, N. Lopez-Martinez, R. De Prado, and H. Walter, Botanical identification of Spanish Echinochloa biotypes with differential responses to quinclorac, in “6th EWRS Mediterranean Symposium,” p. 232, Montpellier, France, 1998. 6. P. W. Michael, Taxonomy and distribution of Echinochloa species with special reference to their occurence as weeds, in “Proceedings, Conference on Weed Control in Rice,” p. 291, IRRI, Los Ban˜os, Philippines, 1981. 7. N. Lopez-Martinez and R. DePrado, Fate of quinclorac in resistant Echinochloa crus-galli, in “Proceedings, Second International Weed Control Congress,” p. 535, Copenhagen, Denmark, 1996. 8. N. Lopez-Martinez, R. H. Shimabukuro, and R. DePrado, Effect of quinclorac on auxin-induced growth, transmembrane proton gradient and ethylene biosynthesis in Echinochloa spp., Aust. J. Plant Physiol. 25, 851 (1998). 9. R. Berghaus and B. Wuerzer, Uptake, translocation and

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

91

metabolism of quinclorac (BAS 514H) in rice and barnyard grass, Proc. 12th Asian-Pac. Weed Sci. Soc. Conf. 133 (1989). G. L. Lamoureux and D. G. Rusness, Quinclorac absorption, translocation, metabolism, and toxicity in leafy spurge (Euphorbia esula), Pestic. Biochem. Physiol. 53, 210 (1995). J. Zawierucha and D. Penner, Differential response of large crabgrass (Digitaria sanguinalis) and goosegrass (Eleusine indica) to quinclorac, WSSA Abstr. 39, 176 (1999). K. Grossmann and F. Scheltrup, Selective induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity is involved in the selectivity of the auxin herbicide quinclorac between barnyard grass and rice, Pestic. Biochem. Physiol. 58, 145 (1997). S. J. Koo, J. C. Neal, and J. M. DiTomaso, Mechanism of action and selectivity of quinclorac in grass roots, Pestic. Biochem. Physiol. 57, 44 (1997). K. Grossmann and J. Kwiatkowski, Selective induction of ethylene and cyanide biosynthesis appears to be involved in the selectivity of the herbicide quinclorac between rice and barnyard grass, J. Plant Physiol. 142, 457 (1993). K. Grossmann, A role for cyanide, derived from ethylene biosynthesis, in the development of stress symptoms, Physiol. Plant. 97, 772 (1996). M. C. C. Lizada and S. F. Yang, A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid, Anal. Biochem. 100, 140 (1979). K. Grossmann and F. Scheltrup, Studies on the mechanism of selectivity of the auxin herbicide quinmerac, Pestic. Sci. 52, 111 (1998). J. L. Lambert, J. Ramasamy, and J. V. Paukstelis, Stable reagents for the colorimetric determination of cyanide by modified Ko¨nig reactions, Anal. Chem. 47, 916 (1975). E. M. Linsmaier and F. Skoog, Organic growth factor requirements of tobacco tissue cultures, Physiol. Plant. 18, 100 (1964). G. Scherer and B. Andre, Stimulation of phospholipase A2 by auxin microsomes from suspension-cultured soybean cells is receptor-mediated and influenced by nucleotides, Planta 191, 515 (1993).