ZEP1 biochemical activity assay

In a 96-well plate, we mixed 180 μl of ZEP1 reaction buffer (35 mM Tris–HCl [pH 8.0], 5 μM zeaxanthin, 0.5 mM NADPH, 5 μM FAD, and 100 mM NaCl). Twenty microliters of recombinant ZEP1-B-His or GST-His were added to the samples to initiate the reaction at room temperature. We collected OD_340 nm of each well as the zero-time-point data. The samples were gently shaken for 5 s every minute to mix them well in the plate reader. After 10 min at room temperature, the OD_340 nm of each well was recorded. For plant ZEP activity, 20 μl of normalized total proteins from leaves at the same position were added to the wells to initiate the reaction. We used a series of NADPH dilutions ranging from 0 to 1.5 mM in the ZEP1 reaction system to build a standard curve. The ZEP1 activity was calculated using the NADPH standard curve with 10 mM Tris–HCl (pH 8.0) as the negative control.