Quantification of Oxidative Stress Markers in Tomato

The oxidative damage and stress tolerance of GLY- and PO-treated S. lycopersicum were quantified based on mRNA markers using real time PCR method. Leaf tissue (100 mg) was homogenized with liquid nitrogen and total RNA was extracted using Trizol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesized following the instructions of the Super Smart cDNA Synthesis Kit (Takara, Otsu, Japan). Real-time quantitative PCR was performed using SYBR Green I master mix (Takara, Otsu, Japan) in a Bio-Rad iCycler iQ5 fluorescence real-time PCR system (Bio-Rad, Hercules, CA, USA). The annealing temperature of the target genes was optimized to 60 °C. The primers for 9-cis-epoxycarotenoid dioxygenase (NCED), superoxide dismutase (SOD1), arginine decarboxylase (ADC), cytochrome P 450 (CYP1A1450), nitrate reductase (NR), Lipooxygenase (LIPO), nitric oxide reductase (NOS) and actin genes are provided in Table 1. To normalize results, the Cq (quantification cycle value) was calculated as the relative expression level of each target gene and the housekeeping gene (Actin) in each sample [65 (link)].