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).