E1 PDH activity assay

Pyruvate dehydrogenase activity was determined by using the PDH Activity Assay Kit (Sigma-Aldrich, MAK183) per the manufacturer’s protocol with adaptation. In short, clean nuclear pellets after nuclear-cytoplasmic fractionation performed on 3-day-old etiolated seedlings from indicated genetic backgrounds were resuspended in 500 μl of PDH assay buffer and later subject to brief sonication shearing and centrifuge to obtain soluble nuclear proteins. Fifty microliters of the cleared nuclear contents was then used to perform the colorimetric enzymatic assay using the Pyruvate dehydrogenase assay kit. The reaction mix was incubated at 37°C. Absorbance at 450 nm was measured by using the Flex Station 3 Multi-Mode Microplate Reader every 1 min and then converted to the amount of NADH (reduced form of nicotinamide adenine dinucleotide; nanomoles per well) generated based on NADH standard curve. Each experimental group was performed with at least three replicates.