Uv 2802pc

The enzymatic activities of the PEPC enzymes were measured as described previously [40 (link), 41 (link)]. Frozen plant tissue was processed in ice-cold glass homogenizers with 500 μl of extraction buffer (50 mM HEPES-KOH pH 7.8, 1 mM EDTA, 0.1% Triton-X, 10 mM dithiothreitol, and polyvinylpolypyrrolidone) and 10 μl of protease inhibitor cocktail (Sigma). The homogenate was briefly centrifuged, and the supernatant was used for the assays. For PEPC, 10 μl of leaf extract was combined with 980 μl of assay buffer (50 mM EPPS-NaOH pH 8, 10 mM MgCl₂, 0.5 mM EDTA, 0.2 mM NADH, 5 mM glucose-6-phosphate 1 mM NaHCO₃ and 1 U ml^− 1 malate dehydrogenase), and the reaction was initiated by the addition of 10 μl of 400 mM PEP. The activity of PEPC was calculated by monitoring the decrease in NADH absorbance at 340 nm with a spectrophotometer (Unico UV-2802PC, USA).

All the nineteen candidate genes’ sequences were obtained on KEGG database. DNAMAN software (Lynnon LLC., CA) was used for protein sequence alignment. Cell density was determined by a UV-visible spectrophotometer (UV-2802PC; Unico, Shanghai, China) via the optical density at 600 nm. After 72 h fermentation, the concentrations of sugars and organic acids concentrations in the samples were analyzed using an Agilent 1260 high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA) equipped with a Bio-Rad HPX-87H column (Bio-Rad Laboratories, Inc., Richmond, CA) with 5 mM H₂SO₄ as the mobile phase (10 μL injection, 0.5 mL/min, 15 °C). Signals were detected by a refractive index (RI) detector.