Submitted to: Weed Biology and Management
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/12/2006
Publication Date: 11/30/2006
Citation: Hirase, K., Hoagland, R.E. 2006. Characterization of aryl acylamidase activity from propanil-resistant barnyardgrass [echinochloa crus-galli (l.) beauv.]. Weed Biology and Management 6:197-203. Interpretive Summary: About 10 years ago, resistance to the herbicide propanil was discovered in an important weed (barnyardgrass) that was initially controlled by propanil. The resistance mechanism in barnyardgrass was due to increased metabolism of propanil by the enzyme aryl acylamidase. We have isolated the enzyme from this resistant weed, and partially characterized it with respect to kinetics, inhibitors, and its relationship to this enzyme in barnyardgrass and rice plants. Levels of the enzyme were higher in resistant barnyardgrass than in the susceptible biotype, which correlates with the degree of resistance found under field conditions. Two herbicides from new chemical classes were found to be potent inhibitors of the enzyme, and this is the first report of the isolation and properties of this enzyme from this propanil-resistant weed. Results are also important because such enzyme inhibition by these herbicides and other compounds could lead to their use, and/or the design of other chemicals, as synergists with propanil to control this herbicide resistant weed.
Technical Abstract: The enzyme aryl acylamidase was isolated and characterized in propanil-sensitive and -resistant barnyardgrass with respect to kinetic parameters, effects of inhibitors, and levels of activity in dark- and light-grown tissues. Generally, enzyme preparations were prepared by homogenizing leaf tissue with phosphate buffer (pH 7.5), dithiothreitol (1 mM), and polyvinylpyrrolidone (0.2%), followed by centrifugation. The supernatants from these preparations were used in standard assays containing enzyme, propanil (1 mM), and phosphate buffer (pH 7.5). Reactions were incubated at 30 deg C and terminated with 20% trichloroacetic acid, followed by centrifugation. The 3,4-dichloroaniline produced was quantitated by adding p-dimethylaminocinnamaldehyde to terminated assay aliquots, and measuring the optical density (A540) after 15 min. The level of extracted enzyme activity in the resistant tissue preparation increased linearly with time over a 5 hr time course, while activity in the sensitive tissue preparation was 2- to 3-fold lower, and the activity tended to decrease at later time points. Apparent Km values were 62 mM and 3 mM for the enzyme activity in sensitive and resistant tissue preparations, respectively. Two herbicides (anilofos and piperophos), previously shown to synergize propanil injury against the resistant biotype, were found to be potent inhibitors of the in vitro aryl acylamidase activity.