ACCase Activity Assays

HetACCase activity was directly monitored in 10-d-old siliques or 21-d-old leaves through the incorporation of ¹⁴CO₂ into acid-stable products as described (Thelen and Ohlrogge, 2002c). Details can be found in the Supplemental Methods. Arabidopsis wild-type Col-0 10-d-old siliques were harvested after 6 h of light exposure, while leaves were grown under constant light conditions and harvested near midday. In each trial, four biological replicates of three siliques or four leaves were assayed. Tissue was pulverized in homogenization buffer (20 mM TES, pH 7.5, 10% glycerol, 5 mM EDTA, 2 mM DTT, 2 mM benzamidine, 2 mM PMSF, and 1% Triton X-100), centrifuged at 10,000g for 15 s, and assayed within 5 min of harvest to minimize loss of hetACCase activity. Assays were performed in the presence of 10 μM haloxyfop to eliminate homomeric ACCase activity. Enzyme activity values for minus acetyl-CoA controls were subtracted from +acetyl-CoA trials to determine the true hetACCase activity levels. Purified recombinant protein was added to assay tube prior to addition of tissue lysate. The K_i value for BADC1 was calculated using the equation fit to the data y = 0.0012x²-0.036x+0.9979, where y equals the normalized ACCase activity at half-maximal inhibition and x equals inhibitor concentration. Maximum inhibition was constrained to the 15 mM BADC1 trial (0.73 normalized ACCase activity).