Relative mRNA Expression Level Analysis

Real-time quantitative reverse transcription-PCR (qRT-PCR) was applied to investigate gene expression patterns. The mRNA levels were measured using SYBR Premix ExTaq^TM II (Perfect Real Time) (TaKaRa, Dalian, China) according to manufacturer’s instructions. qRT-PCR was conducted using ABI 7500 (Applied Biosystems, Foster City, CA, United States). The reactions were performed using biological replicates different from RNA-seq samples in three independent experiments, and three technical replicates were used for each run. Values “Ct” obtained for all genes were normalized to that of an internal control EF1α gene from Arabidopsis. The relative expression level of each gene was calculated using the 2^-ΔΔC_T method (Livak and Schmittgen, 2001). Statistical significances of differences between the sample and control plants were determined by Student’s test. The specific primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, United States) and sequences were listed in Supplementary Table S1.