Quantitative real-time PCR (qRT–PCR) analysis
Citrate synthase (CS) is generally considered as the rate-limiting
enzyme of tricarboxylic acid cycle, whose reduction may lead to an
decreased availability of citrate but increased availability of
oxaloacetate (van Ooyen et al ., 2012). The presence of transcript
levels by qRT-PCR revealed a 3-fold increase in wheat and a 2-fold in
rice for citrate (Si)-synthase by O3 (Fig. 9A). However,
the transcript levels of citrate (Si)-synthase and ATP citrate synthase
were drastically declined by approximately 64% and 90% in soybean
under elevated O3 compared with control, respectively
(Fig. 9A, B). The oxidative
decarboxylation of isocitrate to α-ketoglutarate was catalyzed by
mitochondrial NAD+-specific isocitrate dehydrogenase
(NAD+) (Anderson et al ., 2000), which was
strongly enhanced by O3 in wheat (3.2 fold), rice (1.8
fold) and soybean (6.8 fold) (Fig. 9C). Oxoglutarate dehydrogenase
(succinyl-transferring) is responsible for the conversion of
2-oxoglutarate to succinyl-coenzyme A (Araújo et al ., 2008),
whose expression is highly increased in three crop plants with the
expression being highest (3.1-fold change) in soybean (Fig. 9D). It has
been reported that L-glutamate and oxaloacetate are generated by the
enzyme of aspartate transaminase in the presence of α-ketoglutarate and
L-aspartate (Brauc et al ., 2011; Han et al ., 2011). It was
exhibited that O3 led to a significantly accelerated
induction of aspartate transaminase in three crop plants (Fig. 9E).