Starch degradation and sucrose biosynthesis enzymes analysis
Fresh maize leaves (1.5 g) were ground in an ice-cold mortar containing 4 mL pre-cold 0.1 M citric acid buffer (pH 5.6). The extract was centrifuged at 4 °C, 10000 g, for 5 min. 1 mL was transferred to two tubes separately and diluted 10 times from the supernatant. One tube was used for the measurement of total amylase activity and the other tube was kept at 70 °C for 15 min for α-amylase activity determination. A maltose standard curve was used to calculate the total amylase and α-amylase activities. The β-amylase activity was obtained by subtracting the α-amylase activity from the total amylase activity. 1 U enzyme activity was defined as 1 mg protein degradation starch into 1 mg maltose in 1 min at 37 ℃ and pH 5.6. Starch phosphorylase (SP) was extracted by adding 4 mL extraction buffer (contains 0.1 M sodium succinate; 10% glycerol, 15 mM mercaptoethanol, 1 mM EDTA, 5 mM MgCl2, pH 5.8) to 1.5 g leaf sample. The homogenate was centrifuged at 4 °C, at 16000 g for 10 min. The SP activity was calculated by an inorganic phosphorus (Pi) standard curve. The buffer (0.1 M MES-NaOH, pH 6.5; 5 mM MgCl2; 2 mM EDTA; 50 mM mercaptoethanol; 12.5% (V/V) glycerol) was used for the extraction of starch debranching enzymes (DBEs). The homogenate was centrifuged at 4 °C, 16000 rpm for 10 min. Then 100 μL DBE supernatant and equal volume 50 mM MES-NaOH (pH 6.5) was added in a 2-mL tube and kept at 37 ℃ for 1.5 h. Then 100 μL of 1M Na2CO3 was added to end the reaction, MiniQ water was added in the tube to make the final volume 500 μL. The DBE activity was measured using a 3,5-dinitro salicylic acid method at 525 nm. The activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) were determined as the method by Feng et al. (2019).