Enzyme extraction

Approximately 5.0 g of crown tissue was harvested from each genotype at the two-leaf stage, flash frozen in liquid nitrogen, and stored at − 80 °C. Enzyme extraction and activity assay were modified from previously published methods^19–21. Frozen wheat tissue was powdered in liquid nitrogen. All subsequent extraction steps were carried out on ice or in a cold room to maintain an extract temperature of 4 °C. Ground tissue was transferred to 14.85 mL extraction buffer (100 mM Trizma, 20 mM DTT, 2 mM l-ascorbic acid, 1 mM EDTA, 0.5% w/v PVP-40, 0.5% w/v insoluble PVP-20, and 10% v/v glycerol; pH 8.0) and 150 µL of 100 mM PMSF (dissolved in 100% ethanol). The tissue was homogenized at max speed for 30 s with a homogenizer probe. Homogenate was filtered by hand with Miracloth, followed by centrifugation for 30 min at a speed of 25,000× g. The resulting supernatant was slowly brought to 66% saturation with solid ammonium sulfate and stirred for an hour.

Ammonium sulfate solutions were centrifuged using the same parameters as the previous centrifugation step to precipitate ACCase and similar proteins. After discarding the supernatant, the protein pellets were resuspended with 2.5 mL elution buffer (50 mM tricine, 50 mM KCl, 2.5 mM MgCl₂, and 1 mM DTT; pH = 8.0). The protein extracts were desalted using a gravity protocol with conditioned PD-10 columns. Desalted protein extracts were eluted with 3.5 mL of elution buffer and mixed with 25% v/v glycerol.