- Email: [email protected]
Benzodiazepinediones: A New Class of Photosystem-II-Inhibiting Herbicides
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO.
56, 62–68 (1996)
0059
Benzodiazepinediones: A New Class of Photosystem-II-Inhibiting Herbicides BIJAY K. SINGH,1 IWONA T. SZAMOSI, BRIAN J. DAHLKE, GARY M. KARP, AND DALE L. SHANER American Cyanamid Company, P.O. Box 400, Princeton, New Jersey 08543-0400 Received July 3, 1996; accepted September 20, 1996 Benzodiazepinediones, exemplified by AC 341775, are a new class of herbicides with excellent herbicidal properties. Results from various in vitro and in vivo tests indicate that benzodiazepinediones inhibit photosynthesis by blocking the photosystem II (PSII) electron transport. These compounds do not control triazine-resistant plants; therefore, benzodiazepinediones and triazine herbicides most likely share a common binding site on the D1 protein in PSII. Benzodiazepinediones could be used as molecular probes to understand the structure of PSII. q 1996 Academic Press
INTRODUCTION
cide had symptoms similar to those observed on plants treated with known inhibitors of photosystem II (PSII) (3). These similarities indicated that benzodiazepinediones might be inhibitors of photosynthetic electron transport. Several in vitro and in vivo tests confirmed this idea and conclusively showed that the herbicidal effects of benzodiazepinediones are due to inhibition of electron transport on the reducing side of PSII. Furthermore, benzodiazepinediones and atrazine may share a common binding site on the D1 protein in PSII.
Random screening of compounds has resulted in the discovery of many new chemical classes of herbicidal compounds with diverse mechanisms of action. Through this random screening process, benzodiazepinediones, a new class of compounds with excellent herbicidal activity, were identified (1, 2). AC 341775, one of the most active analogs representing this class of compounds, controls several important weeds and has excellent selectivity in maize. Due to these important features, studies were initiated to understand the mode of action of this class of compounds.
MATERIALS AND METHODS
Greenhouse Evaluation of Herbicidal Activity Plants were grown in a commercial potting mixture. The plant species used and their stage of growth are described under Results and Discussion. AC 341775 was dissolved in 50/ 50 acetone/water with the addition of 0.25% v/v X77 nonionic surfactant. Commercial formulation of atrazine (Aatrex 90 DF) was dissolved in water. The herbicide solutions were sprayed using a laboratory belt sprayer delivering 400 liters/ha spray volume. Visual observations were made at 18 days after application. Herbicidal effects were rated on a scale
In the early greenhouse trials, plants treated with a benzodiazepinedione herbi1 To whom correspondence should be addressed. Fax: (609) 275-5216; E-mail: [email protected].
62 0048-3575/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.
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BENZODIAZEPINEDIONES INHIBIT PHOTOSYSTEM II TABLE 1 Herbicidal Activity of Preemergence Application of AC 341775 on Several Weed Species AC 341775 (g/ha)
Avena fatua Digitaria sanguinalis Echinochloa crus-galli Panicum miliaceum Setaria viridis Triticum aestivum Zea mays Abutilon theophrasti Amaranthus spp. Ambrosia artemisiifolia Chenopodium album Ipomoea hederacea Sesbania exaltata Sinapis arvensis
1000
500
250
125
62.5
31.2
8 9 9 9 9 8 5 9 9 9 9 9 9 9
8 8 8 8 8 7 2 9 9 9 9 9 9 9
6 6 7 6 6 5 0 8 9 8 9 7 9 9
3 3 4 0 3 4 0 5 4 5 9 4 8 8
1 2 1 0 1 0 0 2 0 3 6 2 0 7
0 1 1 0 0 0 0 1 0 2 3 1 0 5
Note. Herbicidal effects were rated on a scale of 0–9, where 0 means no effect and 9 is complete death of the plant.
of 0–9, where 0 means no effect and 9 is complete death of the plant. Cucumber Leaf Disc Leakage Assay This assay was performed according to previously described procedures (4, 5). Discs (5-
mm diameter) were cut with a cork borer from 7-day-old cucumber cotyledons and washed in 1 mM 2-morpholinoethane sulfonic acid (pH 6.5) containing 1% sucrose. Thirty-five discs were placed in 3.5 ml of the wash buffer with or without the test compound. The com-
FIG. 1. Inhibition of photosynthetic electron transport in the ferricyanide reduction assay using spinach thylakoid membranes by AC 341775.
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SINGH ET AL. TABLE 2 Inhibition of Photosynthetic Electron Transport in Different Species by Atrazine and AC 341775 I50 (mM) Species
Atrazine
AC 341775
Avena fatua Panicum miliaceum Setaria viridis Triticum aestivum Zea mays Amaranthus spp. Brassica napus Sesbania exaltata Sinapis arvensis Spinacea oleracea
1.2 1.6 1.2 1.2 1.7 1.2 0.5 3.1 0.8 1.6
3.9 4.9 4.0 4.9 4.5 4.0 1.7 6.2 1.9 6.4
Note. Thylakoid membranes from different species were prepared according to the procedure described under Materials and Methods. Data are presented as the concentration of inhibitor required to cause 50% inhibition of ferricyanide reduction by PSII.
pounds included paraquat (10 mM), atrazine (10 mM), AC 341775 (50 mM), and combinations of different inhibitors as indicated under Results and Discussion. The discs were incubated for 16 hr in dark followed by 8 hr in light. Electrical conductivity of the medium was measured at various times using a conductivity meter. Determination of Amino Acids Soluble amino acid pools were determined in plants according to previously described procedures (6). The meristem regions of the maize seedlings were frozen in liquid nitrogen and then pulverized. Free amino acids were extracted in 0.25 N HCl containing 500 nmol/ ml L-a-amino-b-guanadinopropionic acid as an internal standard. Two milliliters of extraction solution was used for each g of tissue fresh weight. The extract was centrifuged at 25,000g for 15 min. An aliquot of the supernatant (0.25 ml) was loaded on a cation exchange column (AG 50W-X8 from Bio-Rad, Richmond, CA; resin bed volume 4 ml) preequilibrated with 0.01 N HCl. The column was
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washed with 4 ml of 0.01 N HCl and then the amino acids bound with this resin were eluted with 4 1 4-ml aliquots of 9 N ammonium hydroxide. Amino acids eluted from the cation exchange column were freeze dried and then dissolved in Na-S buffer (Beckman, Fullerton, CA). The solution was filtered to remove the particulate matter and the amino acid composition was determined on a Beckman 7300 amino acid analyzer using ninhydrin as the postcolumn derivatization agent. Preparation of Thylakoid Membranes Thylakoid membranes were prepared according to the method of Shimuzu et al. (7). Leaves (50 g) from different plant species were homogenized in an ice-cold blender with 150 ml of 50 mM Tricine – HCl buffer (pH 8) containing 10 mM NaCl and 0.4 M sucrose. The homogenate was filtered through a 100 mM mesh nylon cloth and the filtrate was centrifuged at 2000g for 10 min. The supernatant was discarded and the pellet was resuspended in 10 ml of the homogenization buffer and mixed with an equal volume of glycerol, mixed well, and stored in liquid nitrogen until use. Inhibition of Photosystem II Electron Transport PSII electron transport was measured by following the reduction of potassium ferricyanide by isolated thylakoid membranes from different plant species using the procedure of Shimuzu et al. (7). Inhibition of Photosynthesis Photosynthesis, as monitored by net carbon dioxide uptake, was measured by a LI-6000 portable photosynthesis system according to the manufacturer’s instructions (LI-COR, Inc., Lincoln, NE). RESULTS AND DISCUSSION
Preemergence application of AC 341775 had excellent herbicidal activity on both monocotyledonous and dicotyledonous weed species
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FIG. 2. Net changes in the pools of various amino acids in 2-week-old greenhouse-grown maize seedlings foliarly treated with AC 341775 (2 kg/ha) or pyridate (1 kg/ha). Bars above 0 indicate increases in the free amino acid pools while the bars below 0 indicate decreases in the free amino acid pools.
(Table 1). Plants treated with AC 341775 become chlorotic, then necrotic within a few days. These symptoms are similar to those observed with herbicides that inhibit PSII (e.g., atrazine, diuron, pyridate). This observation led us to evaluate AC 341775 in a number of in vitro and in vivo tests where a comparison was made with various inhibitors of PSII. AC 341775 was found to strongly inhibit photosynthetic electron transport as determined by the in vitro potassium ferricyanide reduction assay using spinach thylakoid membranes (Fig. 1). AC 341775 also inhibited photosynthetic electron transport in a number of different monocot and dicot species (Table 2). The concentration of inhibitor required to cause 50% inhibition of the electron transport in different species was two- to fourfold higher for AC 341775 compared to atrazine. In vitro effects of AC 341775 on photosynthetic electron transport were confirmed in a
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number of direct and indirect in vivo tests. It has been demonstrated that pools of amino acids in plants treated with a test compound can be used to predict the mode of action of a compound (8, 9). Therefore, amino acid pools were determined in 2-week-old maize plants treated with pyridate and AC 341775. Pyridate was used as a standard in this experiment instead of atrazine because this assay uses maize which is not affected by atrazine. Both compounds gave almost identical amino acid profiles (Fig. 2). Both pyridate and AC 341775 caused a reduction in the pool size of Glu. The pools of other amino acids either remained unchanged or increased after treatment with both herbicides. These profiles of amino acid pools were very similar to those observed with other known PSII inhibitors (data for other PSII inhibitors not presented here; 8, 9). This observation indicated that AC 341775 behaves in vivo like other PSII inhibitors.
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FIG. 3. Increase in electrical conductivity of the bathing medium of cucumber cotyledon discs after exposure to paraquat (10 mM), atrazine (10 mM), AC 341775 (50 mM), and combinations of different inhibitors.
Inhibitors of photosynthesis are known to antagonize the herbicidal effects of dipyridinium compounds such as paraquat (10, 11). This antagonistic effect results because the photosynthesis inhibitors reduce the electron supply from the photosynthetic electron transport to photosystem I to paraquat molecules. This inhibition causes diminished superoxide radical generation in plant tissue and thereby reduces paraquat toxicity. This understanding led to the development of an assay to identify inhibitors of photosynthesis (5) as described under Materials and Methods. Paraquat treatment of the leaf discs caused a rapid increase in electrical conductivity of the bathing medium upon exposure to light because of the disruption of cell membranes (Fig. 3). Atrazine and AC 341775 treatment do not cause a significant increase in electrical conductivity of the medium. However, atrazine and AC 341775 significantly reduced cellular damage caused by paraquat as evident from reduced electrical conductivity of the medium when
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they were used in combination with paraquat. These results support the in vitro results that AC 341775 inhibits photosynthetic electron transport before photosystem I. In order to further demonstrate inhibition of photosynthetic electron transport in vivo and its direct effect on inhibition of photosynthesis, net carbon dioxide uptake by intact leaves was determined. Both atrazine and AC 341775 inhibited photosynthesis in a time-dependent manner with nearly complete inhibition of carbon dioxide uptake in 90 min (Fig. 4A). The most convincing evidence came from the tests where AC 341775 was compared with atrazine on a triazine-resistant line of Amaranthus retroflexus in which the resistance is due to an amino acid change in the D1 protein in PSII. Both atrazine and AC 341775 inhibited photosynthetic electron transport in the in vitro PSII assay using thylakoid membranes prepared from a triazine-sensitive biotype of Amaranthus (Fig. 5). In contrast, pho-
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fected by atrazine and AC 341775 in the resistant biotype. These in vitro and in vivo results were supported by the whole plant response observed in these treated plants. While the sensitive biotype was killed by these two herbicides, the resistant biotype was almost unaffected by them (Fig. 4B). Similar results were obtained when a comparison was made between sensitive and triazine-resistant biotypes of other plant species in which resistance to triazines was due an altered binding site of triazines in PSII (data not shown). The results from various in vitro and in vivo tests clearly demonstrate that AC 341775 is an inhibitor of photosynthetic electron transport which causes inhibition of photosynthesis. The binding site of atrazine is the D1 protein
FIG. 4. Inhibition of photosynthesis as measured by net CO2 uptake (A) and plant growth (B) in triazine-sensitive and -resistant biotypes of Amaranthus by atrazine and AC 341775. For the greenhouse evaluation of herbicidal activity, both atrazine and AC 341775 were applied postemergence at 1000 g/ha on 3-week-old seedlings. Herbicidal ratings, as indicated in Table 1, were taken at 18 days after treatment. Data for both net photosynthesis and plant growth are expressed as percentage of untreated control.
tosynthetic electron transport was highly resistant to both compounds in the membranes prepared from an atrazine-resistant biotype of Amaranthus. These in vitro results were confirmed in the in vivo test where photosynthesis was measured as net CO2 uptake. Both atrazine and AC 341775 inhibited photosynthesis almost completely within 90 min in the sensitive biotype of Amaranthus (Fig. 4A). In contrast, photosynthesis was only marginally af-
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FIG. 5. Inhibition of photosynthetic electron transport in triazine-sensitive (l) and -resistant (l) biotypes of Amaranthus by atrazine (A) and AC 341775 (B).
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in PSII and all of the triazine-resistant plants sequenced thus far contain a substitution of Ser264 in this protein (12–15). Since the triazine-resistant weeds are also resistant to AC 341775, the binding site of AC 341775 is most likely the D1 protein in PSII. There are several families of compounds that inhibit PSII (see 16 and references cited therein). The triazine-resistant biotype of Amaranthus is sensitive to many other classes of PSII-inhibiting herbicides (15, 16). Since the triazine-resistant biotype of Amaranthus is also resistant to AC 341775, atrazine and AC 341775 most likely share a common binding site on this protein. A large number of mutants with different types of resistance to various PSII inhibitors have been identified in different microorganisms (15, 16). The large number of inhibitors and the mutants resistant to them have been important factors in understanding the structure and function of PSII. Benzodiazepinediones represent a new class of compounds that inhibit PSII, and the compounds from this family may be useful molecular probes to understand the structure of PSII.
1. M. A. Guaciaro, P. M. Harrington, and G. M. Karp, ‘‘Methods for Controlling Undesirable Plant Species with Benzodiazepinedione Compounds,’’ U.S. Patent 5,438,035, 1995. 2. G. M. Karp, M. A. Guaciaro, P. M. Harrington, M. C. Manfredi, P. Marc, C. L. Ortlip, L. S. Quakenbush, and I. T. Szamosi, Synthesis and herbicidal activity of 1H-1,4-benzodiazepine-2,5-diones, Proceedings of 209th ACS National Meeting, AGRO 048, Anaheim, CA, 1995. 3. F. M. Ashton and A. S. Crafts, ‘‘Mode of Action of Herbicides,’’ Wiley, New York, 1981. 4. W. H. Kenyon, S. O. Duke, and K. C. Vaughn, Sequence of herbicidal effects of acifluorfen on ultra-
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6.
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REFERENCES
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structure and physiology of cucumber cotyledon discs, Pestic. Biochem. Physiol. 24, 240 (1985). D. Yanase and A. Andoh, in ‘‘Target Assays for Modern Herbicides and Related Phytotoxic Compounds’’ (P. Boger and G. Sandmann, Eds.), pp. 257– 262, Lewis Pub., Boca Raton, FL, 1992. B. K. Singh, B. T. Tecle, and D. L. Shaner, Determination of 2-keto acids and amino acids in plant extracts, J. Liq. Chromatogr. 17, 4469 (1994). R. Shimuzu, H. Iwamura, and T. Fujita, Quantitative structure–activity relationships of photosystem II inhibitory anilides and triazines: Topological aspects of their binding to the active site, J. Agric. Food Chem. 36, 1276 (1988). H. Sauter, M. Lauer, and H. Fritsch, in ‘‘Synthesis and Chemistry of Agrochemicals II’’ (D. R. Baker, J. G. Fenyes, and W. K. Moberg, Eds.), pp. 288–299, Am. Chem. Soc., Washington, DC, 1991. B. K. Singh and D. L. Shaner, Changes in free amino acid pools can predict the mode of action of herbicides, Pestic. Sci. 43, 221 (1995). D. Yanase and A. Andoh, Porphyrin synthesis involvement in diphenyl ether-like mode of action of TNPP-ethyl, a novel phenylpyrazole herbicide, Pestic. Biochem. Physiol. 35, 70 (1989). D. Yanase, A. Andoh, and N. Yasudomi, A new simple bioassay to evaluate photosynthetic electrontransport inhibition utilizing paraquat phytotxicity, Pestic. Biochem. Physiol. 38, 92 (1990). J. Hirschberg and L. McIntosh, Molecular basis of herbicide resistance in Amaranthus hybridus, Science 222, 1346 (1983). J. Hirschberg, A. Bleeker, D. Kyle, L. McIntosh, and C. Arntzen, The molecular basis of triazine-herbicide resistance in higher plant chloroplasts, Z. Naturforsch. 39c, 412 (1984). P. Goloubinoff, M. Edelman, and R. B. Hallick, Chloroplast-coded atrazine resistance in Solanum nigrum: psbA loci from susceptible and resistant biotypes are isogenic except for a single codon change, Nucleic Acids Res. 12, 9489 (1984). J. W. Gronwald, in ‘‘Herbicide Resistance in Plants, Biology and Biochemistry’’ (S. B. Powles and J. A. M. Holtum, Eds.), pp. 27–60, CRC Press, Boca Raton, FL, 1994. M. D. Devine, S. O. Duke, and C. Fedtke, ‘‘Physiology of Herbicide Action,’’ Prentice Hall International, Englewood Cliffs, NJ, 1993.
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Benzodiazepinediones: A New Class of Photosystem-II-Inhibiting Herbicides BIJAY K. SINGH,1 IWONA T. SZAMOSI, BRIAN J. DAHLKE, GARY M. KARP, AND DALE L. SHANER American Cyanamid Company, P.O. Box 400, Princeton, New Jersey 08543-0400 Received July 3, 1996; accepted September 20, 1996 Benzodiazepinediones, exemplified by AC 341775, are a new class of herbicides with excellent herbicidal properties. Results from various in vitro and in vivo tests indicate that benzodiazepinediones inhibit photosynthesis by blocking the photosystem II (PSII) electron transport. These compounds do not control triazine-resistant plants; therefore, benzodiazepinediones and triazine herbicides most likely share a common binding site on the D1 protein in PSII. Benzodiazepinediones could be used as molecular probes to understand the structure of PSII. q 1996 Academic Press
INTRODUCTION
cide had symptoms similar to those observed on plants treated with known inhibitors of photosystem II (PSII) (3). These similarities indicated that benzodiazepinediones might be inhibitors of photosynthetic electron transport. Several in vitro and in vivo tests confirmed this idea and conclusively showed that the herbicidal effects of benzodiazepinediones are due to inhibition of electron transport on the reducing side of PSII. Furthermore, benzodiazepinediones and atrazine may share a common binding site on the D1 protein in PSII.
Random screening of compounds has resulted in the discovery of many new chemical classes of herbicidal compounds with diverse mechanisms of action. Through this random screening process, benzodiazepinediones, a new class of compounds with excellent herbicidal activity, were identified (1, 2). AC 341775, one of the most active analogs representing this class of compounds, controls several important weeds and has excellent selectivity in maize. Due to these important features, studies were initiated to understand the mode of action of this class of compounds.
MATERIALS AND METHODS
Greenhouse Evaluation of Herbicidal Activity Plants were grown in a commercial potting mixture. The plant species used and their stage of growth are described under Results and Discussion. AC 341775 was dissolved in 50/ 50 acetone/water with the addition of 0.25% v/v X77 nonionic surfactant. Commercial formulation of atrazine (Aatrex 90 DF) was dissolved in water. The herbicide solutions were sprayed using a laboratory belt sprayer delivering 400 liters/ha spray volume. Visual observations were made at 18 days after application. Herbicidal effects were rated on a scale
In the early greenhouse trials, plants treated with a benzodiazepinedione herbi1 To whom correspondence should be addressed. Fax: (609) 275-5216; E-mail: [email protected].
62 0048-3575/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.
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BENZODIAZEPINEDIONES INHIBIT PHOTOSYSTEM II TABLE 1 Herbicidal Activity of Preemergence Application of AC 341775 on Several Weed Species AC 341775 (g/ha)
Avena fatua Digitaria sanguinalis Echinochloa crus-galli Panicum miliaceum Setaria viridis Triticum aestivum Zea mays Abutilon theophrasti Amaranthus spp. Ambrosia artemisiifolia Chenopodium album Ipomoea hederacea Sesbania exaltata Sinapis arvensis
1000
500
250
125
62.5
31.2
8 9 9 9 9 8 5 9 9 9 9 9 9 9
8 8 8 8 8 7 2 9 9 9 9 9 9 9
6 6 7 6 6 5 0 8 9 8 9 7 9 9
3 3 4 0 3 4 0 5 4 5 9 4 8 8
1 2 1 0 1 0 0 2 0 3 6 2 0 7
0 1 1 0 0 0 0 1 0 2 3 1 0 5
Note. Herbicidal effects were rated on a scale of 0–9, where 0 means no effect and 9 is complete death of the plant.
of 0–9, where 0 means no effect and 9 is complete death of the plant. Cucumber Leaf Disc Leakage Assay This assay was performed according to previously described procedures (4, 5). Discs (5-
mm diameter) were cut with a cork borer from 7-day-old cucumber cotyledons and washed in 1 mM 2-morpholinoethane sulfonic acid (pH 6.5) containing 1% sucrose. Thirty-five discs were placed in 3.5 ml of the wash buffer with or without the test compound. The com-
FIG. 1. Inhibition of photosynthetic electron transport in the ferricyanide reduction assay using spinach thylakoid membranes by AC 341775.
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SINGH ET AL. TABLE 2 Inhibition of Photosynthetic Electron Transport in Different Species by Atrazine and AC 341775 I50 (mM) Species
Atrazine
AC 341775
Avena fatua Panicum miliaceum Setaria viridis Triticum aestivum Zea mays Amaranthus spp. Brassica napus Sesbania exaltata Sinapis arvensis Spinacea oleracea
1.2 1.6 1.2 1.2 1.7 1.2 0.5 3.1 0.8 1.6
3.9 4.9 4.0 4.9 4.5 4.0 1.7 6.2 1.9 6.4
Note. Thylakoid membranes from different species were prepared according to the procedure described under Materials and Methods. Data are presented as the concentration of inhibitor required to cause 50% inhibition of ferricyanide reduction by PSII.
pounds included paraquat (10 mM), atrazine (10 mM), AC 341775 (50 mM), and combinations of different inhibitors as indicated under Results and Discussion. The discs were incubated for 16 hr in dark followed by 8 hr in light. Electrical conductivity of the medium was measured at various times using a conductivity meter. Determination of Amino Acids Soluble amino acid pools were determined in plants according to previously described procedures (6). The meristem regions of the maize seedlings were frozen in liquid nitrogen and then pulverized. Free amino acids were extracted in 0.25 N HCl containing 500 nmol/ ml L-a-amino-b-guanadinopropionic acid as an internal standard. Two milliliters of extraction solution was used for each g of tissue fresh weight. The extract was centrifuged at 25,000g for 15 min. An aliquot of the supernatant (0.25 ml) was loaded on a cation exchange column (AG 50W-X8 from Bio-Rad, Richmond, CA; resin bed volume 4 ml) preequilibrated with 0.01 N HCl. The column was
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washed with 4 ml of 0.01 N HCl and then the amino acids bound with this resin were eluted with 4 1 4-ml aliquots of 9 N ammonium hydroxide. Amino acids eluted from the cation exchange column were freeze dried and then dissolved in Na-S buffer (Beckman, Fullerton, CA). The solution was filtered to remove the particulate matter and the amino acid composition was determined on a Beckman 7300 amino acid analyzer using ninhydrin as the postcolumn derivatization agent. Preparation of Thylakoid Membranes Thylakoid membranes were prepared according to the method of Shimuzu et al. (7). Leaves (50 g) from different plant species were homogenized in an ice-cold blender with 150 ml of 50 mM Tricine – HCl buffer (pH 8) containing 10 mM NaCl and 0.4 M sucrose. The homogenate was filtered through a 100 mM mesh nylon cloth and the filtrate was centrifuged at 2000g for 10 min. The supernatant was discarded and the pellet was resuspended in 10 ml of the homogenization buffer and mixed with an equal volume of glycerol, mixed well, and stored in liquid nitrogen until use. Inhibition of Photosystem II Electron Transport PSII electron transport was measured by following the reduction of potassium ferricyanide by isolated thylakoid membranes from different plant species using the procedure of Shimuzu et al. (7). Inhibition of Photosynthesis Photosynthesis, as monitored by net carbon dioxide uptake, was measured by a LI-6000 portable photosynthesis system according to the manufacturer’s instructions (LI-COR, Inc., Lincoln, NE). RESULTS AND DISCUSSION
Preemergence application of AC 341775 had excellent herbicidal activity on both monocotyledonous and dicotyledonous weed species
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FIG. 2. Net changes in the pools of various amino acids in 2-week-old greenhouse-grown maize seedlings foliarly treated with AC 341775 (2 kg/ha) or pyridate (1 kg/ha). Bars above 0 indicate increases in the free amino acid pools while the bars below 0 indicate decreases in the free amino acid pools.
(Table 1). Plants treated with AC 341775 become chlorotic, then necrotic within a few days. These symptoms are similar to those observed with herbicides that inhibit PSII (e.g., atrazine, diuron, pyridate). This observation led us to evaluate AC 341775 in a number of in vitro and in vivo tests where a comparison was made with various inhibitors of PSII. AC 341775 was found to strongly inhibit photosynthetic electron transport as determined by the in vitro potassium ferricyanide reduction assay using spinach thylakoid membranes (Fig. 1). AC 341775 also inhibited photosynthetic electron transport in a number of different monocot and dicot species (Table 2). The concentration of inhibitor required to cause 50% inhibition of the electron transport in different species was two- to fourfold higher for AC 341775 compared to atrazine. In vitro effects of AC 341775 on photosynthetic electron transport were confirmed in a
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number of direct and indirect in vivo tests. It has been demonstrated that pools of amino acids in plants treated with a test compound can be used to predict the mode of action of a compound (8, 9). Therefore, amino acid pools were determined in 2-week-old maize plants treated with pyridate and AC 341775. Pyridate was used as a standard in this experiment instead of atrazine because this assay uses maize which is not affected by atrazine. Both compounds gave almost identical amino acid profiles (Fig. 2). Both pyridate and AC 341775 caused a reduction in the pool size of Glu. The pools of other amino acids either remained unchanged or increased after treatment with both herbicides. These profiles of amino acid pools were very similar to those observed with other known PSII inhibitors (data for other PSII inhibitors not presented here; 8, 9). This observation indicated that AC 341775 behaves in vivo like other PSII inhibitors.
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FIG. 3. Increase in electrical conductivity of the bathing medium of cucumber cotyledon discs after exposure to paraquat (10 mM), atrazine (10 mM), AC 341775 (50 mM), and combinations of different inhibitors.
Inhibitors of photosynthesis are known to antagonize the herbicidal effects of dipyridinium compounds such as paraquat (10, 11). This antagonistic effect results because the photosynthesis inhibitors reduce the electron supply from the photosynthetic electron transport to photosystem I to paraquat molecules. This inhibition causes diminished superoxide radical generation in plant tissue and thereby reduces paraquat toxicity. This understanding led to the development of an assay to identify inhibitors of photosynthesis (5) as described under Materials and Methods. Paraquat treatment of the leaf discs caused a rapid increase in electrical conductivity of the bathing medium upon exposure to light because of the disruption of cell membranes (Fig. 3). Atrazine and AC 341775 treatment do not cause a significant increase in electrical conductivity of the medium. However, atrazine and AC 341775 significantly reduced cellular damage caused by paraquat as evident from reduced electrical conductivity of the medium when
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they were used in combination with paraquat. These results support the in vitro results that AC 341775 inhibits photosynthetic electron transport before photosystem I. In order to further demonstrate inhibition of photosynthetic electron transport in vivo and its direct effect on inhibition of photosynthesis, net carbon dioxide uptake by intact leaves was determined. Both atrazine and AC 341775 inhibited photosynthesis in a time-dependent manner with nearly complete inhibition of carbon dioxide uptake in 90 min (Fig. 4A). The most convincing evidence came from the tests where AC 341775 was compared with atrazine on a triazine-resistant line of Amaranthus retroflexus in which the resistance is due to an amino acid change in the D1 protein in PSII. Both atrazine and AC 341775 inhibited photosynthetic electron transport in the in vitro PSII assay using thylakoid membranes prepared from a triazine-sensitive biotype of Amaranthus (Fig. 5). In contrast, pho-
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fected by atrazine and AC 341775 in the resistant biotype. These in vitro and in vivo results were supported by the whole plant response observed in these treated plants. While the sensitive biotype was killed by these two herbicides, the resistant biotype was almost unaffected by them (Fig. 4B). Similar results were obtained when a comparison was made between sensitive and triazine-resistant biotypes of other plant species in which resistance to triazines was due an altered binding site of triazines in PSII (data not shown). The results from various in vitro and in vivo tests clearly demonstrate that AC 341775 is an inhibitor of photosynthetic electron transport which causes inhibition of photosynthesis. The binding site of atrazine is the D1 protein
FIG. 4. Inhibition of photosynthesis as measured by net CO2 uptake (A) and plant growth (B) in triazine-sensitive and -resistant biotypes of Amaranthus by atrazine and AC 341775. For the greenhouse evaluation of herbicidal activity, both atrazine and AC 341775 were applied postemergence at 1000 g/ha on 3-week-old seedlings. Herbicidal ratings, as indicated in Table 1, were taken at 18 days after treatment. Data for both net photosynthesis and plant growth are expressed as percentage of untreated control.
tosynthetic electron transport was highly resistant to both compounds in the membranes prepared from an atrazine-resistant biotype of Amaranthus. These in vitro results were confirmed in the in vivo test where photosynthesis was measured as net CO2 uptake. Both atrazine and AC 341775 inhibited photosynthesis almost completely within 90 min in the sensitive biotype of Amaranthus (Fig. 4A). In contrast, photosynthesis was only marginally af-
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FIG. 5. Inhibition of photosynthetic electron transport in triazine-sensitive (l) and -resistant (l) biotypes of Amaranthus by atrazine (A) and AC 341775 (B).
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in PSII and all of the triazine-resistant plants sequenced thus far contain a substitution of Ser264 in this protein (12–15). Since the triazine-resistant weeds are also resistant to AC 341775, the binding site of AC 341775 is most likely the D1 protein in PSII. There are several families of compounds that inhibit PSII (see 16 and references cited therein). The triazine-resistant biotype of Amaranthus is sensitive to many other classes of PSII-inhibiting herbicides (15, 16). Since the triazine-resistant biotype of Amaranthus is also resistant to AC 341775, atrazine and AC 341775 most likely share a common binding site on this protein. A large number of mutants with different types of resistance to various PSII inhibitors have been identified in different microorganisms (15, 16). The large number of inhibitors and the mutants resistant to them have been important factors in understanding the structure and function of PSII. Benzodiazepinediones represent a new class of compounds that inhibit PSII, and the compounds from this family may be useful molecular probes to understand the structure of PSII.
1. M. A. Guaciaro, P. M. Harrington, and G. M. Karp, ‘‘Methods for Controlling Undesirable Plant Species with Benzodiazepinedione Compounds,’’ U.S. Patent 5,438,035, 1995. 2. G. M. Karp, M. A. Guaciaro, P. M. Harrington, M. C. Manfredi, P. Marc, C. L. Ortlip, L. S. Quakenbush, and I. T. Szamosi, Synthesis and herbicidal activity of 1H-1,4-benzodiazepine-2,5-diones, Proceedings of 209th ACS National Meeting, AGRO 048, Anaheim, CA, 1995. 3. F. M. Ashton and A. S. Crafts, ‘‘Mode of Action of Herbicides,’’ Wiley, New York, 1981. 4. W. H. Kenyon, S. O. Duke, and K. C. Vaughn, Sequence of herbicidal effects of acifluorfen on ultra-
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