Quantification of ACCase Activity in Digitaria ciliaris

After growing to the four-leaf stage, seedlings of four D. ciliaris var. chrysoblephara populations were sprayed with the recommended dose of metamifop (120 g a.i.ha⁻¹). Seedlings were collected from each population at 0, 2, 12, 24, 48, and 72 h after treatment, respectively. ACCase catalyzes the transformation of Acetyl-CoA, NaHCO₃, and ATP to generate Malonyl-CoA, ADP, and inorganic phosphorus. The interaction of molybdenum blue and phosphate can generate products with characteristic absorption peaks at 660 nm. Thus, ACCase activity was determined based on inorganic phosphorus levels using the ammonium molybdate method. Specifically, ACCase activity was quantified using an ACCase activity assay kit (Biobox, China) and a microplate spectrophotometer (Agilent BioTek Epoch2, USA). Crude enzyme was prepared by adding 1 mL of extracting solution to 0.1 g of leaf tissue. The enzymatic reactions and phosphate quantification were then conducted based on the manufacturer’s instructions. Absorbance values at 660 nm were determined for experimental reactions, in addition to those for negative controls, blank controls, and to establish a standard curve. Three biological replicates and three technical replicates were used for each sample.
The unit of ACCase activity was calculated based on sample mass, as defined by the amount of 1 μmol of inorganic phosphorus generated for 1 g tissue over 1 h. Specifically, ACCase activity was calculated with the following equation:
In the formula, V_total is the total volume of the enzymatic reaction (0.1 mL), V_sample is the volume of added sample (0.01 mL), V_{total sample} is the volume of extracting solution (1 mL), T is the time of enzymatic reaction (0.5 h), and W is the fresh weight of sample (0.1 g).