Nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) contents were determined following the method of Hu et al. (2016) (link). Nitrite reductase (NiR) activity was assayed according to the method previously described (Seith et al., 1994 (link)).
The activity of C-metabolizing enzymes was measured as follows. For the preparation of enzyme solution, 1 g of the fruit sample was ground into an ice bath in a precooled mortar; 100 mol·l-1 of 5 ml Tris–HCl (pH 7.0) buffer, containing 2% glycol, 2 mol·l-1 EDTA, 5 mol·l-1 MgCl2, 2% PVPP, 2% bovine serum protein (BSA), and 5 mmol·L-1 DTT, was added for fractional times. 3 ml of the supernatant was put into a dialysis bag after centrifugation at 4°C and 10,000 r·min-1 for 20 min. The extraction buffer diluted five times (removing PVPP) was used for dialysis for 15 to 24 h at low temperature (2°C–4°C). The enzyme solution after dialysis was used for determination of various enzyme activities. Sorbitol dehydrogenase (SDH) activity was determined as described by Rufly and Huber (1983) (link). Sorbitol oxidase (SOX) activity was determined as described by Yamaki and Asakura (1991) (link). Sucrose synthase decomposition direction activity (SS-c) was determined, as described by Huber (1983) (link). Sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were determined as described by Xu et al. (2012) (link). The activities of acid invertase (AI) and neutral invertase (NI) were determined as described by Merlo and Passera (1991) (link).
The activity of C-metabolizing enzymes was measured as follows. For the preparation of enzyme solution, 1 g of the fruit sample was ground into an ice bath in a precooled mortar; 100 mol·l-1 of 5 ml Tris–HCl (pH 7.0) buffer, containing 2% glycol, 2 mol·l-1 EDTA, 5 mol·l-1 MgCl2, 2% PVPP, 2% bovine serum protein (BSA), and 5 mmol·L-1 DTT, was added for fractional times. 3 ml of the supernatant was put into a dialysis bag after centrifugation at 4°C and 10,000 r·min-1 for 20 min. The extraction buffer diluted five times (removing PVPP) was used for dialysis for 15 to 24 h at low temperature (2°C–4°C). The enzyme solution after dialysis was used for determination of various enzyme activities. Sorbitol dehydrogenase (SDH) activity was determined as described by Rufly and Huber (1983) (link). Sorbitol oxidase (SOX) activity was determined as described by Yamaki and Asakura (1991) (link). Sucrose synthase decomposition direction activity (SS-c) was determined, as described by Huber (1983) (link). Sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were determined as described by Xu et al. (2012) (link). The activities of acid invertase (AI) and neutral invertase (NI) were determined as described by Merlo and Passera (1991) (link).
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