In vivo activity of ALS

In order to know how the gametocide amidosulfuron affect plant ALS proteins in vivo, the activities of ALS enzymes in mature leaf and young flower bud (length ≤ 3 mm) were assayed0, 1, 4, 7, and 13 days after treatment (DAT). The in vivo assay ALS activity, which was represented by accumulation of the substrate acetolactate, can monitor the activity inhibition by the ALS-inhibiting herbicide that is absorbed and even partially degraded by plant (Gerwick et al., 1993; Simpson et al., 1995). It had advantages over in vitro assay because the latter method is used to compare the activity of ALS proteins, isolated from different genotypes, reacting with exogenous substrate, and is not appropriate in cases of metabolic detoxification of the herbicide, unsteady supply of substrate, or altered expression of the ALS genes in the treated plants. In brief, the plants in a treatment were sprayed with 0.5 mol/L of 1,1-cyclopropanedicarboxylate (CPCA), a specific inhibitor of ketol-acid reductoisomerase (KARI) subsequent to ALS, to accumulate ALS enzyme substrate acetolactate. After 24 h, leaf or flower bud samples were collected from the CPCA treated plants and 1 g sample was extracted with 5 mL of 0.05 M sodium phosphate buffer (pH 7.2) and centrifuged 10 min at 8,000 × g. 0.5 mL supernatant was incubated at 60°C for 10 min. 0.1 mL of 1 M sulfuric acid was added to stop the catalysis of ALS enzyme and the incubation was maintained for 30 min to generate acetoin. Then 1 mL of 0.5% (W/V) creatine (prepared in 2 M NaOH) and 1 mL of 5% (W/V) alpha naphthol (in 2 M NaOH) were added and kept at 37°C for 30 min to transform acetoin into a red compound. Supernatant was obtained after an instant centrifugation and detected by using a spectrophotometer. Absorbance of supernatant was measured at 530 nm (A530) and then ALS activity was calculated as A530 per hour by 1 g fresh sample. Each samples had three replicates and Student's t-test was used for comparisons of the same tissues collected on the same day.