Submitted to: Weed Biology and Management
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/20/2001
Publication Date: 2/1/2002
Citation: Hirase, K., Molin, W.T. 2002. Measuring cysteine biosynthesis activity from serine in extracts from sorghum, corn and grassy weeds, and their metolachlor susceptibility. Weed Biology and Management 2:52-59. Interpretive Summary: This paper describes the assay procedure for measuring the synthesis of cysteine, a sulfur containing amino acid, from serine and sulfide. This reaction is important because the cysteine formed is a precursor to glutathione, which is an important component of herbicide detoxification reactions. The procedure was used to characterize cysteine biosynthesis from several weeds and crops. The cysteine biosynthesis capacity was correlated to the susceptibility to metolachlor, a herbicide detoxified by glutathione conjugation. A seed protectant enhanced the level of cysteine biosynthesis activity. This research shows that cysteine biosynthesis is an important part of the mechanisms contributing to herbicide selectivity and tolerance.
Technical Abstract: In vitro assay procedures for measuring the activity of cysteine biosyn- thesis from serine (CBS) were developed using crude extracts from sorghum shoots. CBS is a coupled reaction in which serine acetyltransferase catalyzes the production of )-acetylserine (OAS) from serine and acetyl-CoA and then cysteine synthase catalyzes the synthesis of cysteine from OAS and dsulfide. The reaction was initiated by addtion of serine, acetyl-CoA, and sulfide. CBS activity was dependent on acetyl-CoA concentrations (up to 1.5 mM), serine (at least up to 20 mM) and sulfide (up to 0.25 mM), respectively. CBS activity was proportional to the protein concentration in the reaction mixture below 0.4 mg/ml. The reaction rate was 6.6 nmol/min/mg protein during the first 5 min, but increased to 45.6 nmol/min/mg protein between 30 and 45 min after reaction initiation. Sorghum had the highest CBS total activity (222.4 nmol/min/g fresh weight), and large crabgrass had dthe lowest CBS total activity (4.7 nmol/min/g fresh weight) when CBS activity in shoots was extracted from sorghum, corn, johnsongrass, barnyardgrass, goosegrass, green foxtail and large crabgrass. Similar results were obtained for CBS specific activity (nmol/min/mg protein). There was no correlation between total CBS activity and susceptibility to metolachlor, however, when corn was excluded, a correlation of R**2=0.690 was found. Flurazole seed treatment (1.25 g/kg seed) conferred metolachlor resistance by sorghum, and enhanced total CBS activity and nonprotein thiol content by 27% and 61%, respectively. From these results, the difference in CBS activity partially contributes to metolachlor selectivity among certain grass species, andto the safening action of flurazole by increasing thiol content.