PMI in vivo assay

The specific activity of PMI in vivo was determined according to the coupled enzymatic assay procedure, with a slight modification⁵³. Young leaf tissues (500 mg) were extracted in 400 μL of ice-cold Tris-HCl (50 mM, pH 7.5) containing 1% protease inhibitor cocktail (Sigma), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT) and 1 mM polyvinylpyrrolidone (PVP). One hundred microliters of crude protein extract was added to the substrate solution consisting of 75 μL of NADP (13.5 mM), 1 μL of PGI (1 kU/mL), and 0.5 μL of GDPDH (1 kU/mL) and incubated for 30 min at 37 °C to remove any endogenous D-M6P. The reaction was initiated by adding 20 μL of D-M6P (50 mM), and the ▵A₃₄₀ was recorded for 60 min to calculate the activities.