Sybr green

The leaf samples were frozen in liquid nitrogen immediately after sampling and ground with TissueLyser II (Qiagen, Hilden, Germany). Total RNA was extracted using RNAiso Plus according to the manufacturer’s protocol (TakaraBio, Kyoto, Japan). MMLV Reverse Transcriptase (Promega, WI, USA) and oligo (dT) primers were used for cDNA synthesis. We carried out a qRT–PCR analysis using a Rotor-Gene Q instrument system (Qiagen, Hiden, Germany), and the synthesized cDNAs were amplified using 2× Prime Q-Master mix with SYBR Green (GeNet Bio, Nonsan-Si, Korea).
For the normalization of the amplified transcripts, we used the rice ubiquitin 5 gene as an internal control of the qRT–PCR analysis (OsUbi5, LOC_Os01g22490) [51 (link),52 (link)]. In addition, Late Elongated Hypocotyl (OsLHY, LOC_Os08g06110) was used as a positive control for the investigation of diurnal rhythms. The quality of samples under abiotic stress was monitored using OsbZIP23 (LOC_Os02g52780) [53 (link)] for drought and salt and OsNAC6 for cold (LOC_Os01g66120) [54 (link)] as positive controls (Figure S1). All primers used in these qRT–PCR analyses are summarized in Table S2.

Real-time PCR analysis was carried out with the Stratagene Agilent Real-time PCR system (Agilent Technologies, CA, USA) for total RNA extraction according to the manufacturer’s guidelines at cycling conditions at 95 °C for 5 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The primers shown in Table 1 and SYBR Green (GeNetbio) were used for the quantification of the mRNA of iNOS and COX-2 and the endogenous control β-actin with the fold change in target mRNAs calculated using the comparative ΔΔCt method [68 (link)].