RNA extraction

For total RNA extraction, 0.2 g of frozen leaf material was ground in lysing buffer (2% CTAB, 2% PVP K-30, 100 mM Tris-HCl pH 8.0, 2 M NaCl, 25 mM EDTA, β-MeOH (200 µl/10ml) added in prior to use (Chang, Puryear, & Cairney, 1993). Thoroughly vortexed solution was then extracted twice with equal volume of acid phenol-chloroform-isoamyl-OH (ph 4.5). Prior to precipitation, 160 µl of 10M LiCl was added and samples were kept on ice overnight, followed by 30 min centrifugation (10000 rpm) in +4 °C. Pellets were dissolved in 500 µl of 65 °C SSTE (1M NaCl, 0.5% SDS, 10 mmTris-HCl pH 8.0, 1mM EDTA) and RNA was extracted twice with chloroform-Isoamyl alcohol (24∶1). After EtOH precipitation and 70% wash, the pellets were dissolved in 40 µl H2O and RNA quantity and quality were checked using NanoDrop (Thermo Fischer Scientific). Potential contamination of genomic DNA in the RNA samples was removed using DNase I (Thermo Fischer Scientific) and samples were then reverse-transcribed to cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions.