4.9. Determination of NAD+, NADH, ADP, ATP Contents, and Enzyme Activities Related to TCA Cycle

0.5 g of soybean roots were used for the determination of NAD⁺, NADH, ADP, ATP contents, and enzyme activities related to the TCA cycle. The NAD⁺ and NADH contents were determined following the method of Wang et al. [86]. ADP and ATP contents were assayed using ELISA kits (Shanghai Jiwei Biological Technology Co., Ltd., Shanghai, China) according to the method as described by the manufacturer’s instructions.

PDH activity was determined according to the method of Millar et al. [87]. Root samples were extracted in an enzyme extraction buffer (80 mM tetrasodium pyrophosphate (pH 7.5), 0.3 M mannitol, 10 mM K₃PO₄, 25 mM cysteine, 2 mM EGTA, 1% PVP and 1% BSA). After centrifugation 2000× g for 5 min, the supernatant was obtained and continued centrifuged 15,000× g for 20 min, then resuspended the precipitation with 1 mL of resuspension buffer (10 mM TES-KOH (pH 7.5), 0.3 M mannitol, and 0.1% BSA). After centrifugation at 30,000× g for 45 min, the supernatant was the crude enzyme solution of PDH. To determinate the PDH activity, a 100 μL crude enzyme solution was added to the reaction buffer (75 mM Hepes-NaOH (pH 7.5), 10 mM NAD⁺, 10 mM MgCl₂, 10 mM cysteine, 0.2 mM CoA, 2 mM thiamine pyrophosphate) and incubated at 25 °C for 15 min. After the 20 mM, sodium pyruvate was added, the changes of absorbance value at 340 nm within 3 min were recorded. The activities of CS, IDH, Fum, and MDH were determined according to the method of Jenner et al. [88]. α-KGDH was performed according to the method of Pekovich et al. [89]. Root samples were extracted in an enzyme extraction buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 0.01% Triton X-100 and 0.02% sodium deoxycholate. After centrifugation at 10,000× g for 10 min at 4 °C, the supernatant was used for the determination of α-KGDH. The α-KGDH activity was determined following the method of Pekovich et al. [89]. Briefly, a 400 μL crude enzyme solution was added to the reaction buffer (50 mM MOPS (pH 8.0), 10 mM NAD⁺, 2 mM MgCl₂, 0.16 mM CoA, 1.2 mM CaCl₂, 0.04 mM rotenone and 0.5% Triton X-100) and incubated at 25 °C for 15 min. After the 20 mM α-ketoglutarate was added, the changes of absorbance value at 340 nm within 3 min were recorded. SDH was determined following the method of Schirawski and Unden [90].