Extraction and RT-PCR Analysis of Total RNA

Total RNA was extracted from approximately 200 mg of freshly sampled leaf tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Any genomic contamination was removed before cDNA synthesis using RNase-free DNase I (TaKaRa, Dalian, China), and according to the manufacturer’s protocols. Nucleic acid quality was estimated by visual analysis on 1.2% agarose gel electrophoresis, according to standard procedures [93] . RNA concentrations were measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE, USA) and only RNA samples with an A260/A280 ratio in the range 1.8–2.0 were used, in order to minimise the effects of PCR inhibitors. All RNA samples were stored at −80°C.
The first strand of cDNA was synthesised from 1.5 µg total RNA with the M-MLV reverse transcriptase and oligo (dT)₁₅ primer (Promega, Madison, WI, USA) according to user instructions. In brief, total RNA samples were denatured at 95°C for 3 minutes in the presence of 10 pM oligo (dT)₁₅ primer and then quickly cooled on ice. M-MLV reverse transcriptase and other reaction components were added to the samples. These were then incubated for 10 minutes at 37°C (primer annealing), followed by 90 minutes at 42°C and finally 10 minutes at 70°C to inactivate the enzyme. Reverse transcription (RT) negative controls, without the inclusion of the reverse transcriptase enzyme, were performed in parallel to test for the presence of genomic DNA contamination in RNA samples. Amplification was then conducted for all genes using RT-PCR, followed by assessment on a 4% agarose gel. No visible amplification was detected in any of the control samples (Figure S4B).