Imidazolinones: Potent Inhibitors of Acetohydroxyacid Synthase

Mark  Stidham

Imidazolinones: Potent Inhibitors of Acetohydroxyacid Synthase

Mark Stidham

1984, PLANT PHYSIOLOGY

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The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K, for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides.

Plant Physiol. (1984) 76, 545-546 0032-0889/84/76/0545/02/$0 1.00/0 Short Communication Imidazolinones POTENT INHIBITORS OF ACETOHYDROXYACID SYNTHASE Received for publication May 7, 1984 and in revised form August 7, 1984 DALE L. SHANER*, PAUL C. ANDERSON, AND MARK A. STIDHAM American Cyanamid Company, P. 0. Box 400, Princeton, New Jersey 08540 (D. L. S., M. A. S.); and Molecular Genetics, Inc., 10320 Bren Road East, Minnetonka, Minnesota 55343 (P. C. A.) ABSTRACT The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K, for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides. The imidazolinones are a new chemical class of herbicides discovered and being developed by American Cyanamid Company (see structures in Table I). These herbicides kill both monocotyledonous and dicotyledonous species, and selectivity is achieved by differential metabolism of the compounds to nonherbicidal forms (7, 9). Death of the whole plant can require several weeks. The meristematic tissues die first, followed by slow necrosis of the mature tissue. The precise mechanism of action of many herbicides are unknown. The imidazolinones are somewhat unusual in that a biochemical pathway has been identified which is inhibited by these herbicides. The imidazolinones interfere with the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine (10). Corn tissue grown in suspension culture and treated with AC 243,997 (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]nicotinic acid) showed changes in the free amino acid levels (10). Most of the amino acids either increased or remained constant, but the levels of valine, leucine, and isoleucine decreased. Exogenously supplying these three amino acids to corn tissue cultures and seedlings prevented the phytotoxic effects of AC 243,997 (10). All three of these amino acids were necessary for maximal protection to occur. There are four enzymes that catalyze biosynthetic steps leading to valine, leucine, and isoleucine. Acetohydroxyacid synthase (EC 4.1.3.18) is the first enzyme in the biosynthesis ofvaline and leucine and the second enzyme in the biosynthesis of isoleucine. Acetohydroxyacid synthase is feedback inhibited cooperatively by valine and leucine in higher plants (4). Thus, it is a key controlling point for the levels of the branched chain amino acids and is a prime site for inhibition by a xenobiotic. The imidazolinones were found to be potent inhibitors of acetohydroxyacid synthase and this may explain why the levels of valine, leucine, and isoleucine decreased after treatment with the imidazolinones. MATERIALS AND METHODS Chemicals. Analytical grade imidazolinones were synthesized at the American Cyanamid Agricultural Research Center, Princeton, NJ. Acetoin was obtained from Eastman Kodak. Plant Material. Corn (Zea mays L. Pioneer var 3547) seeds were planted in sand and watered with 0.2 mm CaC12. The plants were grown in darkness at 28°C for 3 d. The roots and shoots from the etiolated seedlings were used. Acetohydroxyacid Synthase Assay. The procedure used was a modification of one used by B. J. Miflin (5). Roots and coleoptiles of the corn seedlings were homogenized in an extraction buffer (50 mm K-phosphate buffer (pH 7.5),5 mM EDTA, 5 mm MgCl2, 1 mM valine, 1 mM leucine). The homogenate was filtered through eight layers of cheese cloth and centrifuged at 14,000g for 15 min. The supernatant fraction was brought to 65% saturation with respect to (NH4)jSO4 and allowed to stand 1 h on ice. Then the mixture was centrifuged at 14,000g for 15 min and the supernatant was discarded. The (NH4)SO4 precipitated pellet was redissolved in 50 mM K-phosphate buffer (pH 7.5), 1 mm EDTA, and 0.1 M NaCl. This solution was passed through a Sephadex G-25 column equilibrated with the same buffer. The eluate containing the desalted protein was collected and used for the assay procedure. The assay solution contained 20 mM pyruvate, 0.32 mm thiamine pyrophosphate, 0.5 mm MnSO4,20 mM K-phosphate buffer (pH 7, 0.5 ml), and 0.4 ml of enzyme solution. Inhibitors were added in 0.1-ml aliquots to bring the final volume to 1 ml. This mixture was incubated at 37C for I h after which time the reaction was stopped with 0.2 ml of 30% H2SO4. The reaction tubes were assayed for acetolactate by decarboxylation at 600C for 15 min and subsequent measurement of the acetoin formed by the method of Westerfield (11). The imidazolinones were tested for inhibition of acetohydroxyacid synthase activity over a range of 4.6 x I0O- to 1 x I0O M. The apparent Ki values were calculated using a Dixon Plot (8). RESULTS AND DISCUSSION Three different imidazolinones were compared for their inhibitory action on acetohydroxyacid synthase. All three were potent inhibitors of the enzyme (Table I). We have found that these imidazolinones also have excellent herbicidal activity and that their phytotoxic effects can be reversed by exogenous applications of valine, leucine, and isoleucine (10). The inhibition of acetohydroxyacid synthase by AC 243,997 was uncompetitive as shown in the Hanes-Woolf plot (Fig. 1). This indicates that this imidazolinone binds to the enzymepyruvate complex. The acetohydroxyacid synthase we extracted from corn was cooperatively inhibited by valine and leucine. The Downloaded from on June 6, 2020 545- Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 76, 1984 SHANER ET AL. 546 Table I. K, Values for ImidazolinonesforAcetohydroxyacid Synthase Extractedfrom Corn Seedings H N O0 R N CH(CH A. CH35 AC Number R <COOH 243,997 COOH 222,164 CO~COH 252,214 I I I . I I I I AC 243,997 (9). Jensen (4) and Clowes (3) have shown that the rate of protein synthesis is highest in the root tip region. Furthermore, observations by Oaks (6) showed that in excised maize embryos an adequate supply of amino acids is required in addition to a carbohydrate for a normal increase in protein nitrogen. Oaks (6) also showed that inhibition ofleucine synthesis by externally supplying this amino acid caused a change in the internal pool size of leucine in maize root tips. If the imidazolinones inhibit the synthesis of valine, leucine, and isoleucine in vivo, there may be a rapid decrease in the pool sizes of these amino acids. This could cause a decrease in protein synthesis. A lower rate of protein synthesis, in turn, could cause a slow down in the rate of cell division, and eventually death of the cell. Mature tissue has larger pools of amino acids as well as protein reserves that can be catabolized for amino acids. Thus, it should take longer for the phytotoxic effect of the imidazolinones to become apparent in mature tissue, as was observed. The inhibition of amino acid synthesis is a relatively new mechanism of action of herbicides. Until recently, glyphosate was the only other herbicide that was known to interfere with amino acid biosynthesis. Glyphosate inhibits the biosynthesis of aromatic amino acids (1). The symptomatology of glyphosate has some similarities to the imidazolinones in that the meristematic tissue dies first followed by slow necrosis of the mature tissue. More recently, Chaleff and Mauvais (2) reported that acetohydroxyacid synthase is inhibited by the sulfonyl area herbicides. It appears that the sulfonyl ureas and the imidazolinones may have the same mechanism of action. The effectiveness of these types of herbicides indicates that amino acid biosynthesis is a target for the disruption of plant metabolism and could be a site of action of future herbicides. LITERATURE CITED S/V 1 A CPYRUVATE] a a a A 16 20 24 28 32 (mM) FIG. 1. Inhibition of acetohydroxyacid synthase by AC 243,997. Activities were measured in 20 mm K-phosphate buffer (pH 7.5) at 37-C with different concentrations of pyruvate. The levels of AC 243,997 used were 0 (0), 3.1 pM (0), 6.2 JLM (X), 12.5 pM (U), 25 AM (0). properties were similar to those described by Miflin (5) for acetohydroxyacid synthase extracted from barley shoots. The inhibition of acetohydroxyacid synthase by the imidazolinones could explain the herbicidal effects of these compounds. These herbicides kill plants very slowly and the first symptoms appear in the meristematic tissue. In corn root tips, the level of the soluble proteins decreased within 24 h after application of 1. AMRHEIN N, B DEUS, P GEHRKE, HC STEINRUCHEN 1980 The site of the inhibition of the shikimate pathway by glyphosate. II. Interference of glyphosate with chorismate formation in vivo and in vitro. Plant Physiol 66: 830-834 2. CHALEFF RS, Cl MAUVAIS 1984 Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224: 1443-1445 3. CLOwEs FAL 1958 Protein synthesis in root meristems J Exp Bot 7: 307-312 4. JENSEN WA 1957 The incorporation of C'4-adenine and C'4-phenylalanine by developing root tip cells. Proc Nail Acad Sci USA 43: 1038-1046 5. MIFLIN BJ 1971 Cooperative feedback of barley acetohydroxyacid synthase by leucine, isoleucine and valine. Arch Biochem Biophys 146: 542-550 6. OAKS A 1965 The effect ofleucine on the biosynthesis of leucine in maize root tips. Plant Physiol 40: 149-155 7. ORWICK PL, PA MARC, K UMEDA, DL SHANER, M Los, DR CIARLANTE 1983 AC 252,214-A new broad spectrum herbicide for soybeans: greenhouse studies. Proc South Weed Sci Soc 36: 90 8. SEGEL IH 1976 Biochemical Calculations, Ed 2. John Wiley and Sons, New York 9. SHANER DL, P ROBSON, PD Simcox, DR CIARLANTE 1983 Absorption, translocation and metabolism of AC 252,214 in soybeans, cocklebur and velvetleaf. Proc South Weed Sci Soc 36: 92 10. SHANER DL, PC ANDERSON, M RENDER, MA STIDHAM, PL ORWICK 1984 Physiological responses of corn (Zea mays) to ARSENAL herbicide. Proc South Weed Sci Soc. In press I 1. WESTERFIELD WW 1945 A colorimetric determination ofblood acetoin. J Biol Chem 161: 495-502 Downloaded from on June 6, 2020 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved.

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