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).