Quantitative Gene Expression Analysis

Total RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and reverse transcription using the SuperScript system (Invitrogen) and oligo(dT) primer was carried out according to the manufacturer’s instructions. Real-time PCR was performed to measure gene expression was carried out as previously described (Jaffé et al., 2012 (link); Nazri et al., 2017 (link)) using SYBR Green (Finnzymes) and an Opticon DNA Engine Continuous Fluorescence Detector (GRI Ltd.). PCR was performed using specific forward and reverse primers for each gene (Supporting information Table S1) at 95 °C for 10 min followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. All gene expression analysis was performed with at least three independent biological replicates and the reactions were set up in triplicate for each sample. All data were standardized by normalizing to SAND or Yellow-Leaf-Specific gene8 (YLS8) expression (Remans et al., 2008 (link); Jaffé et al., 2012 (link)) and analysed using Opticon Monitor III software (Biorad). Quantification of the relative transcript levels was performed using the comparative Ct (threshold cycle) method.