RNA Extraction and qRT-PCR Analysis

Total RNA from root tissues of 30 seedlings was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. DNA-free total RNA (2 µg) from different treatments was used for first-strand cDNA synthesis in a 20-µL reaction volume containing 2.5 units of avian myeloblastosis virus reverse transcriptase XL (TaKaRa) and oligo dT primer.
Real-time quantitative RT-PCR reactions were performed with Mastercycler realplex² real-time PCR system (Eppendorf, Hamburg, Germany) using the SYBR Premix Ex Taq (TaKaRa) according to the user manual. The cDNA was amplified using primers (Table S1). The expression levels of the genes are presented as values relative to the corresponding control samples under the indicated conditions, with normalization of data to the geometic average of two internal control genes MSC27 and Actin2[46] (link).