Quantification of MKK gene expression

Total RNA was extracted from the collected samples with a method modified from the previously described [48 ]. First strand cDNA synthesis was carried out using 1 μg total RNA and a PrimeScritpt RT reagent Kit (TaKaRa, Japan) following the manufacturer’s instructions. Reverse transcription - quantitative PCR (qRT-PCR) was performed as previously described [7 , 33 (link)]. The gene-specific primers used in the qRT-PCR analysis of MKK genes were designed using Beacon Designer 7.0 software (Premier Biosoft International, USA) and targeted the 3′ UTR of each of the genes and designed to generate approximately 200 bp products (Additional files 8: Table S5). Grapevine actin gene was selected as an internal control to normalize the total amounts of cDNA present in each reaction. The relative expression levels of the target genes were assessed using the 2^-ΔΔCt method, where ΔΔCt = (Ct_{target gene} - Ct_actin) _treatment - (Ct_{target gene} - Ct_actin) _control [7 ]. Each sample comprised three biological replicates and the analysis represented the results of three independent experiments.