Supporting information S1
Assay mixture composition for enzyme activity measurement
Determination of NAD-MDH activity (Kulichikhin, Chirkova & Fagerstedt,
2009). The assay mixture contained 0.1 M HEPES-KOH (pH 7.5), 5 mM MgSO4, 0.2
mM NADH, 2 mM oxaloacetic acid (OAA), and 10 μl of enzyme extract in a total
volume of 1 ml. The reaction was started by the addition of OAA.
Determination of PEPC activity (Kulichikhin , Chirkova & Fagerstedt, 2009).
The reaction medium contained 100 mM HEPES-KOH (pH 8.0), 5 mM MgSO4, 5 mM
KHCO3,
5
mM
DTT,
5
mM
glucose-6-phosphate,
5
mM
trisodium
phosphoenolpyruvate, 0.2 mM NADH, 5 EU/ml of MDH from pig heart (Oriental Yeast
Co. Ltd., Tokyo, Japan), and 100 μl of enzyme extract in a total volume of 1 ml.
Determination of NADP-ME activity (Kulichikhin, Chirkova & Fagerstedt,
2009). The reaction medium contained 100 mM HEPES-KOH (pH 7.5), 5 mM MgSO4,
0.5 mM NADP+, 5 mM L-(+)-malate, and 100 µl of enzyme extract in a total volume of
1 ml.
Determination of PPDK activity. (Moons, Valke & Van Montagu, 1998, with
some modifications). Tris-HCl buffer in the original protocol was replaced by HEPESKOH, MgCl2 was replaced by MgSO4, and NaHCO3 was replaced by KHCO3. The
modified assay mixture contained 100 mM HEPES-KOH (pH 7.5), 10 mM MgSO4, 0.1
mM Na2EDTA, 50 mM KHCO3, 1.25 mM sodium pyruvate, 1.25 mM ATP, 5 mM
DTT, 2.5 mM K2HPO4, 0.2 mM NADH, 2 U/ml PEPC from maize leaves (Wako Pure
Chemical Industries Inc., Osaka, Japan), 2U/ml of MDH from pig heart (Oriental Yeast
Co. Ltd., Tokyo, Japan), and 100 to 140 µl of root extract in a total volume of 1 ml.